U.S. patent number RE41,365 [Application Number 12/378,811] was granted by the patent office on 2010-06-01 for nested oligonucleotides containing a hairpin for nucleic acid amplification.
This patent grant is currently assigned to Alexion Pharmaceuticals, Inc.. Invention is credited to Katherine S. Bowdish, Shana Frederickson, Ying-Chi Lin, Toshiaki Maruyama, John McWhirter.
United States Patent |
RE41,365 |
Bowdish , et al. |
June 1, 2010 |
Nested oligonucleotides containing a hairpin for nucleic acid
amplification
Abstract
Templates that are engineered to contain a predetermined
sequence and a hairpin structure are provided by a nested
oligonucleotide extension reaction. The engineered template allows
Single Primer Amplification (SPA) to amplify a target sequence
within the engineered template. In particularly useful embodiments,
the target sequences from the engineered templates are cloned into
expression vehicles to provide a library of polypeptides or
proteins, such as, for example, an antibody library.
Inventors: |
Bowdish; Katherine S. (Del Mar,
CA), Frederickson; Shana (Solana Beach, CA), McWhirter;
John (Chula Vista, CA), Maruyama; Toshiaki (La Jolla,
CA), Lin; Ying-Chi (San Diego, CA) |
Assignee: |
Alexion Pharmaceuticals, Inc.
(Cheshire, CT)
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Family
ID: |
26944185 |
Appl.
No.: |
12/378,811 |
Filed: |
February 18, 2009 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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60254669 |
Dec 11, 2000 |
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60323400 |
Sep 19, 2001 |
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Reissue of: |
10014012 |
Dec 10, 2001 |
06919189 |
Jul 19, 2005 |
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Current U.S.
Class: |
435/91.2;
435/6.16; 536/24.33; 536/24.32; 536/24.3; 536/23.1; 435/91.1 |
Current CPC
Class: |
C12Q
1/6844 (20130101); C12Q 1/6844 (20130101); C12Q
2525/301 (20130101) |
Current International
Class: |
C12P
19/34 (20060101); C12Q 1/68 (20060101); C07H
21/04 (20060101) |
Field of
Search: |
;435/91.2,91.21,6
;536/23.1,24.33,24.3,24.32 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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258017 |
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Mar 1988 |
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EP |
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50424 |
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Sep 1988 |
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EP |
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84796 |
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Feb 1990 |
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EP |
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237362 |
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Mar 1992 |
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EP |
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201184 |
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Dec 1992 |
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EP |
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368684 |
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Mar 1994 |
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EP |
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WO-9014430 |
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Nov 1990 |
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WO |
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WO-97/04131 |
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Feb 1997 |
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WO |
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Other References
Mullis et al. Cold spring Harbor Symp. Quant. Biol. 51:263-273;
(1986). cited by other .
Patel et al., Proc.Natl. Acad. Sci. USA 93:2969-2974 (1996). cited
by other.
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Primary Examiner: Wilder; Cynthia B
Attorney, Agent or Firm: Ropes & Gray LLP
Parent Case Text
RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application
No. 60/254,669 filed December 11, 2000 and to U.S. Provisional
Application No. 60/323,400 filed Sep. 19, 2001. The disclosures of
both these Provisional Applications are incorporated herein in
their entirety by this reference.
Claims
We claim:
1. A method of amplifying nucleic acid comprising the steps of: a)
annealing a primer to a template nucleic acid sequence, the primer
having a first portion which anneals to the template and a second
portion of predetermined sequence; b) synthesizing a polynucleotide
that anneals to and is complementary to the portion of the template
adjacent to the location at which the first portion of the primer
anneals to the template, the polynucleotide having a first end and
a second end, wherein the first end incorporates the primer, c)
separating the polynucleotide synthesized in step (b) from the
template; d) annealing a nested oligonucleotide to the second end
of the polynucleotide synthesized in step (b), the nested
oligonucleotide having a first portion that anneals to the second
end of the polynucleotide, and a second portion having a hairpin
structure; e) extending the polynucleotide synthesized in step (b)
to provide an extended polynucleotide comprising a portion that is
complementary to the hairpin structure and a terminal portion that
is complementary to the predetermined sequence; and f) amplifying
the extended polynucleotide using a single primer having the
predetermined sequence.
2. A method as in claim 1 further comprising the step of providing
a nucleic acid template by annealing a restriction oligonucleotide
to a nucleic acid strand to form a double stranded portion and
digesting the nucleic acid strand at the double stranded
portion.
3. A method as in claim 1 wherein the template encodes an
immunoglobulin molecule or fragment thereof.
4. A method as in claim 1 wherein the template is selected from the
group consisting of full length or truncated mRNA, DNA and
cDNA.
5. A method as in claim 1 wherein the nucleic acid being amplified
includes a target sequence encoding a polypeptide.
6. A method as in claim 5 wherein the target sequence encodes an
immunoglobulin molecule or fragment thereof.
7. A method as in claim 5 further comprising the step of digesting
the extended polynucleotide to isolate the target sequence.
8. A method as in claim 7 further comprising the step of ligating
the isolated target sequence into an expression vector.
9. A method as in claim 8 further comprising the steps of
transforming a host cell with the expression vector and expressing
the polypeptide encoded by the target sequence.
10. A method of amplifying nucleic acid comprising the steps of: a)
annealing a primer and a boundary oligonucleotide to a template
nucleic acid sequence, the primer having a first portion which
anneals to the template and a second portion of predetermined
sequence; b) synthesizing a polynucleotide that anneals to and is
complementary to the portion of the template between the location
at which the first portion of the primer anneals to the template
and the portion of the template to which the boundary
oligonucleotide anneals, the polynucleotide having a first end and
a second end, wherein the first end incorporates the primer; c)
separating the polynucleotide synthesized in step (b) from the
template; d) annealing a nested oligonucleotide to the second end
of the polynucleotide synthesized in step (b), the nested
oligonucleotide having a first portion that anneals to the second
end of the polynucleotide and a second portion having a hairpin
structure; e) extending the polynucleotide synthesized in step (b)
to provide an extended polynucleotide comprising a portion that is
complementary to the hairpin structure and a terminal portion that
is complementary to the predetermined sequence; and f) amplifying
the extended polynucleotide using a single primer having the
predetermined sequence.
11. A method as in claim 10 further comprising the step of
providing a nucleic acid template by generating first strand cDNA
from mRNA.
12. A method as in claim 10 wherein the template is selected from
the group consisting of full length or truncated mRNA, DNA and
cDNA.
13. A method as in claim 10 wherein the extended polynucleotide
includes a target sequence encoding a polypeptide.
14. A method as in claim 10 wherein the extended polynucleotide
encodes an immunoglobulin molecule or fragment thereof.
15. A method as in claim 14 wherein the target sequence encodes an
immunoglobulin molecule or fragment thereof.
16. A method as in claim 14 further comprising the step of
digesting the extended polynucleotide to isolate the target
sequence.
17. A method as in claim 16 further comprising the step of ligating
the isolated target sequence into an expression vector.
18. A method as in claim 17 further comprising the steps of
transforming a host cell with the expression vector and expressing
the polypeptide encoded by the target sequence.
19. A method of amplifying nucleic acid comprising the steps of: a)
annealing an oligo dT primer and a boundary oligonucleotide to an
mRNA template; b) synthesizing a polynucleotide that anneals to and
is complementary to the portion of the template between the
location at which the first portion of the primer anneals to the
template and the portion of the template to which the boundary
oligonucleotide anneals, the polynucleotide having a first end and
a second end, wherein the first end incorporates the primer; c)
separating the polynucleotide synthesized in step (b) from the
template; d) annealing a nested oligonucleotide to the second end
of the polynucleotide synthesized in step (b), the nested
oligonucleotide having a first portion that anneals to the second
end of the polynucleotide, and a second portion having a hairpin
structure; e) extending the polynucleotide synthesized in step (b)
to provide an extended polynucleotide comprising a portion that is
complementary to the hairpin structure and a poly A terminal
portion; and f) amplifying the extended polynucleotide using a
single primer.
20. A method as in claim 19 further comprising the step of
providing a nucleic acid template by generating first strand cDNA
from mRNA.
21. A method as in claim 19 wherein the template is selected from
the group consisting of full length or truncated mRNA, DNA and
cDNA.
22. A method as in claim 19 wherein the extended polynucleotide
includes a target sequence encoding a polypeptide.
23. A method as in claim 19 wherein the extended polynucleotide
encodes an immunoglobulin molecule or fragment thereof.
24. A method as in claim 22 wherein the target sequence encodes an
immunoglobulin molecule or fragment thereof.
25. A method as in claim 22 further comprising the step of
digesting the extended polynucleotide to isolate the target
sequence.
26. A method as in claim 25 further comprising the step of ligating
the isolated target sequence into an expression vector.
27. A method as in claim 26 further comprising the steps of
transforming a host cell with the expression vector and expressing
the polypeptide encoded by the target sequence.
28. A method of amplifying a nucleic acid strand comprising the
steps of: a) providing a nucleic acid strand having i) a
predetermined sequence engineered onto a first end thereof, ii) a
sequence complementary to the predetermined sequence, and iii) a
hairpin structure therebetween; and b) contacting the engineered
nucleic acid strand with a primer containing at least a portion of
the predetermined sequence in the presence of a polymerase and
nucleotides under conditions suitable for polymerization of the
nucleotides, thereby producing a complementary nucleic acid
strand.
29. A method as in claim 28 further comprising the steps of:
digesting the complementary nucleic acid strand to isolate a target
nucleic acid sequence contained therein; ligating the target
nucleic acid sequence into an expression vector; transforming a
host organism with the expression vector; and expressing a
polypeptide or protein encoded by the target sequence.
30. A method of amplifying a family of related nucleic acid
sequences to build a complex library of polypeptides encoded by the
sequences, the method comprising: a) annealing a primer to a family
of related nucleic acid sequence templates, the primer having a
first portion which anneals to the templates and a second portion
of predetermined sequence; b) synthesizing polynucleotides that
anneal to and are complementary to the portion of the templates
adjacent to the location at which the first portion of the primer
anneals to the templates, the polynucleotides having a first end
and a second end, wherein the first end incorporates the primer; c)
separating the polynucleotides synthesized in step (b) from the
templates; d) annealing a nested oligonucleotide to the second end
of the polynucleotides synthesized in step (b), the nested
oligonucleotide having a first portion that anneals to the second
end of the polynucleotides, and a second portion having a hairpin
structure; e) extending the polynucleotides synthesized in step (b)
to provide an extended polynucleotide comprising a portion that is
complementary to the hairpin structure and a terminal portion that
is complementary to the predetermined sequence; and f) amplifying
the extended polynucleotides using a single primer having the
predetermined sequence.
31. A method as in claim 1, wherein steps a), b) and c) are
repeated from 15 to 25 times prior to annealing the nested
oligonucleotide.
32. A method as in claim 10, wherein steps a), b) and c) are
repeated from 15 to 25 times prior to annealing the nested
oligonucleotide.
33. A method as in claim 19, wherein steps a), b) and c) are
repeated from 15 to 25 times prior to annealing the nested
oligonucleotide.
34. A method as in claim 30, wherein steps a), b) and c) are
repeated from 15 to 25 times prior to annealing the nested
oligonucleotide.
35. A method as in claim 1 wherein the first end of the
polynucleotide is the 5' end.
36. A method as in claim 1 wherein the first end of the
polynucleotide is the 5' end.
37. A method as in claim 19 wherein the first end of the
polynucleotide is the 5' end.
38. A method as in claim 1 wherein the first end of the nucleic
acid strand is the 5' end.
Description
TECHNICAL FIELD
This disclosure relates to engineered templates useful for
amplification of a target nucleic acid sequence. More specifically,
templates which are engineered to contain a predetermined sequence
and a hairpin structure are provided by a nested oligonucleotide
extension reaction. The engineered templates allow Single Primer
Amplification (SPA) to amplify a target sequence within the
engineered template. In particularly useful embodiments, the target
sequences from the engineered templates are cloned into expression
vehicles to provide a library of polypeptides or proteins, such as,
for example, an antibody library.
BACKGROUND OF RELATED ART
Methods for nucleic acid amplification and detection of
amplification products assist in the detection, identification,
quantification, isolation and sequence analysis of nucleic acid
sequences. Nucleic acid amplification is an important step in the
construction of libraries from related genes such as, for example,
antibodies. These libraries can be screened for antibodies having
specific, desirable activities. Nucleic acid analysis is important
for detection and identification of pathogens, detection of gene
alteration leading to defined phenotypes, diagnosis of genetic
diseases or the susceptibility to a disease, assessment of gene
expression in development, disease and in response to defined
stimuli, as well as the various genome projects. Other applications
of nucleic acid amplification method include the detection of rare
cells, detection of pathogens, and the detection of altered gene
expression in malignancy, and the like. Nucleic acid amplification
is also useful for qualitative analysis (such as, for example, the
detection of the presence of defined nucleic acid sequences) and
quantification of defined gene sequences (useful, for example, in
assessment of the amount of pathogenic sequences as well as the
determination of gene multiplication or deletion, and cell
transformation from normal to malignant cell type, etc.). The
detection of sequence alterations in a nucleic acid sequence is
important for the detection of mutant genotypes, as relevant for
genetic analysis, the detection of mutations leading to drug
resistance, pharmacogenomics, etc.
There are many variations of nucleic acid amplification, for
example, exponential amplification, linked linear amplification,
ligation-based amplification, and transcription-based
amplification. One example of exponential nucleic acid
amplification method is polymerase chain reaction (PCR) which has
been disclosed in numerous publications. See, for example, Mullis
et al. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986);
Mullis K. EP 201,184; Mullis et al. U.S. Pat. No. 4,582,788; Erlich
et al. EP 50,424, EP 84,796, EP 258,017, EP 237,362; and Saiki R.
et al. U.S. Pat. No. 4,683,194. In fact, the polymerase chain
reaction (PCR) is the most commonly used target amplification
method. PCR is based on multiple cycles of denaturation,
hybridization of two different oligonucleotide primers, each to
opposite strand of the target strands, and primer extension by a
nucleotide polymerase to produce multiple double stranded copies of
the target sequence.
Amplification methods that employ a single primer, have also been
disclosed. See, for example, U.S. Pat. Nos. 5,508,178; 5,595,891;
5,683,879; 5,130,238; and 5,679,512. The primer can be a DNA/RNA
chimeric primer, as disclosed in U.S. Pat. No. 5,744,308.
Some amplification methods use template switching oligonucleotides
(TSOs) and blocking oligonucleotides. For example, a template
switch amplification in which chimeric DNA primer are utilized is
disclosed in U.S. Pat. Nos. 5,679,512; 5,962,272; 6,251,639; and by
Patel et al. Proc. Natl. Acad. Sci. U.S.A. 93:2969-2974 (1996).
However the previously described target amplification methods have
several drawbacks. For example, the transcription base
amplification methods, such as Nucleic Acid Sequence Based
Amplification (NASBA) and transcription mediated amplification
(TMA), are limited by the need for incorporation of the polymerase
promoter sequence into the amplification product by a primer, a
process prone to result in non-specific amplification. Another
example of a drawback of the current amplification methods is the
requirement of two binding events which may have optimal binding at
different temperatures. This combination of factors results in
increased likelihood of mis-priming and resultant amplification of
sequences other than the target sequence. Therefore, there is a
need for improved nucleic acid amplification methods that overcome
these drawbacks. The invention provided herein fulfills this need
and provides additional benefits.
SUMMARY
A method of amplifying nucleic acid has been discovered which
includes the steps of a) annealing a primer to a template nucleic
acid sequence, the primer having a first portion which anneals to
the template and a second portion of predetermined sequence; b)
synthesizing a polynucleotide that anneals to and is complementary
to the portion of the template between the location at which the
first portion of the primer anneals to the template and the end of
the template, the polynucleotide having a first end and a second
end, wherein the first end incorporates the primer; c) separating
the polynucleotide synthesized in step (b) from the template; d)
annealing a nested oligonucleotide to the second end of the
polynucleotide synthesized in step (b), the nested oligonucleotide
having a first portion that anneals to the second end of the
polynucleotide, and a second portion having a hairpin structure; e)
extending the polynucleotide synthesized in step (b) to provide a
portion that is complementary to the hairpin structure and a
terminal portion that is complementary to the predetermined
sequence; and f) amplifying the extended polynucleotide using a
single primer having the predetermined sequence.
In an alternative embodiment, the method of amplifying nucleic acid
includes the steps of a) annealing a primer and a boundary
oligonucleotide to a template nucleic acid sequence, the primer
having a first portion which anneals to the template and a second
portion of predetermined sequence; b) synthesizing a polynucleotide
that anneals to and is complementary to the portion of the template
between the location at which the first portion of the primer
anneals to the template and the portion of the template to which
the boundary oligonucleotide anneals, the polynucleotide having a
first end and a second end, wherein the first end incorporates the
primer; c) separating the polynucleotide synthesized in step (b)
from the template; d) annealing a nested oligonucleotide to the
second end of the polynucleotide synthesized in step (b), the
nested oligonucleotide having a first portion that anneals to the
second end of the polynucleotide and a second portion having a
hairpin structure; e) extending the polynucleotide synthesized in
step (b) to provide a portion that is complementary to the hairpin
structure and a terminal portion that is complementary to the
predetermined sequence; and f) amplifying the extended
polynucleotide using a single primer having the predetermined
sequence.
In yet another embodiment, the method of amplifying nucleic acid
includes the steps of a) annealing an oligo dT primer and a
boundary oligonucleotide to an mRNA template; b) synthesizing a
polynucleotide that anneals to and is complementary to the portion
of the template between the location at which the first portion of
the primer anneals to the template and the portion of the template
to which the boundary oligonucleotide anneals, the polynucleotide
having a first end and a second end, wherein the first end
incorporates the primer; c) separating the polynucleotide
synthesized in step (b) from the template; d) annealing a nested
oligonucleotide to the second end of the polynucleotide synthesized
in step (b), the nested oligonucleotide having a first portion that
anneals to the second end of the polynucleotide, and a second
portion having a hairpin structure; e) extending the polynucleotide
synthesized in step (b) to provide an extended polynucleotide that
includes a portion that is complementary to the hairpin structure
and a poly A terminal portion; and f) amplifying the extended
polynucleotide using a single primer.
In another aspect an engineered nucleic acid strand is disclosed
which has a predetermined sequence at a first end thereof, a
sequence complementary to the predetermined sequence at the other
end thereof, and a hairpin structure therebetween.
In yet another aspect, a method of amplifying a nucleic acid strand
has been discovered which includes the steps of providing an
engineered nucleic acid strand having a predetermined sequence at a
first end thereof, a sequence complementary to the predetermined
sequence at the other end thereof and a hairpin structure
therebetween, and contacting the engineered nucleic acid strand
with a primer containing at least a portion of the predetermined
sequence in the presence of a polymerase and nucleotides under
conditions suitable for polymerization of the nucleotides.
Once the engineered nucleic acid is amplified a desired number of
times, restriction sites can be used to digest the strand so that
the target nucleic acid sequence can be ligated into a suitable
expression vector. The vector may then be used to transform an
appropriate host organism using standard methods to produce the
polypeptide or protein encoded by the target sequence. In
particularly useful embodiments, the techniques described herein
are used to amplify a family of related sequences to build a
complex library, such as, for example, an antibody library.
BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 is a schematic illustration of a primer and boundary oligo
annealed to a template;
FIG. 2A is a schematic illustration of a restriction oligo annealed
to a nucleic acid strand;
FIG. 2B is a schematic illustration of a primer annealed to a
template that has a shortened 5' end;
FIG. 3 is a schematic illustration of a nested oligo having a
hairpin structure annealed to a newly synthesized nucleic acid
strand;
FIG. 4A is a schematic illustration of an engineered template in
accordance with this disclosure; and
FIG. 4B is a schematic illustration of an engineered template in
accordance with an alternative embodiment.
FIGS. 5A-5C is a chart showing the sequences of clones produced in
Example 4.
FIGS. 6A-6C is a chart showing the sequences of clones produced in
Example 7.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present disclosure provides a method of amplifying a target
nucleic acid sequence. In particularly useful embodiments, the
target nucleic acid sequence is a gene encoding a polypeptide or
protein. The disclosure also describes how the products of the
amplification may be cloned and expressed in suitable expression
systems. In particularly useful embodiments, the techniques
described herein are used to amplify a family of related sequences
to build a complex library, such as, for example, an antibody
library.
The target nucleic acid sequence is exponentially amplified through
a process that involves only a single primer. The ability to employ
a single primer (i.e., without the need for both forward and
reverse primers each having different sequences) is achieved by
engineering a strand of nucleic acid that contains the target
sequence to be amplified. The engineered strand of nucleic acid
(sometimes referred to herein as the "engineered template") is
prepared from two templates; namely, 1) a starting material that is
a natural or synthetic nucleic acid (e.g., RNA, DNA or cDNA)
containing the sequence to be amplified and 2) a nested
oligonucleotide that provides a hairpin structure. The starting
material can be used directly as the original template, or,
alternatively, a strand complementary to the starting material can
be prepared and used as the original template. The nested
oligonucleotide is used as a template to extend the nucleotide
sequence of the original template during creation of the engineered
strand of nucleic acid. The engineered strand of nucleic acid is
created from the original template by a series of manipulations
that result in the presence of a predetermined sequence at one end
thereof and a hairpin structure. It is these two features that
allow amplification using only a single primer.
Any nucleic acid, in purified or nonpurified form, can be utilized
as the starting material for the processes described herein
provided it contains or is suspected of containing the target
nucleic acid sequence to be amplified. Thus, the starting material
employed in the process may be, for example, DNA or RNA, including
messenger RNA, which DNA or RNA may be single stranded or double
stranded. In addition, a DNA-RNA hybrid which contains one strand
of each may be utilized. A mixture of any of these nucleic acids
may also be employed, or the nucleic acids produced from a previous
amplification reaction herein using the same or different primers
may be utilized. The target nucleic acid sequence to be amplified
may be a fraction of a larger molecule or can be present initially
as a discrete molecule. The starting nucleic acid may contain more
than one desired target nucleic acid sequence which may be the same
or different. Therefore, the present process may be useful not only
for producing large amounts of one target nucleic acid sequence,
but also for amplifying simultaneously more than one different
target nucleic acid sequence located on the same or different
nucleic acid molecules.
The nucleic acids may be obtained from any source, for example:
genomic or cDNA libraries, plasmids, cloned DNA or RNA, or from
natural DNA or RNA from any source, including bacteria, yeast,
viruses, and higher organisms such as plants or animals. The
nucleic acid can be naturally occurring or synthetic, either
totally or in part. Techniques for obtaining and producing the
nucleic acids used in the present processes are well known to those
skilled in the art. If the nucleic acid contains two strands, it is
necessary to separate the strands of the nucleic acid before it can
be used as the original template, either as a separate step or
simultaneously with the synthesis of the primer extension products.
Additionally, if the starting material is first strand DNA, second
strand DNA may advantageously be created by processes within the
purview of those skilled in the art and used as the original
template from which the engineered template is created.
First strand cDNA and mRNA are particularly useful as the original
template for the present methods. Suitable methods for generating
DNA templates are known to and readily selected by those skilled in
the art. In one embodiment, 1.sup.st strand cDNA is synthesized in
a reaction where reverse transcriptase catalyzes the synthesis of
DNA complementary to any RNA starting material in the presence of
an oligodeoxynucleotide primer and the four deoxynucleoside
triphosphates, dATP, dGTP, dCTP, and TTP. The reaction is initiated
by the non-covalent bonding of the oligo-deoxynucleotide primer to
the 3' end of mRNA followed by stepwise addition of the appropriate
deoxynucleotides as determined by base pairing relationships with
the mRNA nucleotide sequence, to the 3' end of the growing chain.
As those skilled in the art will appreciate, all mRNA in a sample
can be used to generate first strand cDNA through the annealing of
oligo dT to the poly A tail of the mRNA.
Once the original template is obtained, a primer 20 and a boundary
oligonucleotide 30 arc annealed to the original template 10. (See
FIG. 1.) A strand of nucleic acid complementary to the portion of
the original template beginning at the 3' end of the primer up to
about the 5' end of the boundary oligonucleotide is
polymerized.
The primer 20 that is annealed to the original template includes a
portion 25 that anneals to the original template and optionally a
portion 22 of predetermined sequence that preferably does not
anneal to the template, and optionally a restriction site 23
between portions 22 and 25. Thus, for example, where the original
template is mRNA, a portion having a predetermined sequence that
does not anneal to the template is not needed, but rather the
primer can be any gene-specific internal sequence of the mRNA or
oligo dT which will anneal to the unique poly A tail of the
mRNA.
The primer anneals to the original template adjacent to the target
sequence 12 to be amplified. It is contemplated that the primer can
anneal to the original template upstream of the target sequence (or
downstream in the case, e.g., of an mRNA original template) to be
amplified, or that the primer may overlap the beginning of the
target sequence 12 to be amplified as shown in FIG. 1. The
predetermined sequence of portion 22 of the primer is selected so
as to provide a sequence to which the single primer used during the
amplification process can hybridize as described in detail below.
Preferably, the predetermined sequence is not native in the
original template and does not anneal to the original template, as
shown in FIG. 1. Optionally, the predetermined sequence may include
a restriction site useful for insertion of a portion of the
engineered template into an expression vector as described more
fully hereinbelow.
The boundary oligonucleotide 30 that is annealed to the original
template serves to terminate polymerization of the nucleic acid.
Any oligonucleotide capable of terminating nucleic acid
polymerization may be utilized as the boundary oligonucleotide 30.
In a preferred embodiment the boundary oligonucleotide includes a
first portion 35 that anneals to the original template 10 and a
second portion 32 that is not susceptible to an extension reaction.
Techniques to prevent the boundary oligo from acting as a site for
extension are within the purview of one skilled in the art. By way
of example, portion 32 of the boundary oligo 30 may be designed so
that it does not anneal to the original template 10 as shown in
FIG. 1. In such embodiments, the boundary oligonucleotide 30
prevents further polymerization but does not serve as a primer for
nucleic acid synthesis because the 3' end thereof does not
hybridize with the original template 10. Alternatively, the 3' end
of the boundary oligo 30 might be designed to include locked
nucleic acid to achieve the same effect. Locked nucleic acid is
disclosed for example in WO 99/14226, the contents of which are
incorporated herein by reference. Those skilled in the art will
envision other ways of ensuring that no extension of the 3' end of
the boundary oligo occurs.
Primers and oligonucleotides described herein may be synthesized
using established methods for oligonucleotide synthesis which are
well known in the art. Oligonucleotides, including primers of the
present invention include linear oligomers of natural or modified
monomers or linkages, such as deoxyribonucleotides,
ribonucleotides, and the like, which are capable of specifically
binding to a target polynucleotide by way of a regular pattern of
monomer-to monomer interactions such as Watson-Crick base pairing.
Usually monomers are linked by phosphodiester bonds or their
analogs to form oligonucleotides ranging in size from a few
monomeric units e.g., 3-4, to several tens of monomeric units. A
primer is typically single-stranded, but may be double-stranded.
Primers are typically deoxyribonucleic acids, but a wide variety of
synthetic and naturally occurring primers known in the art may be
useful for the methods of the present disclosure. A primer is
complementary to the template to which it is designed to hybridize
to serve as a site for the initiation of synthesis, but need not
reflect the exact sequence of the template. In such a case,
specific hybridization of the primer to the template depends on the
stringency of the hybridization conditions. Primers may be labeled
with, e.g., chromogenic, radioactive, or fluorescent moieties and
used as detectable moieties.
Polymerization of nucleic acid can be achieved using methods known
to those skilled in the art. Polymerization is generally achieved
enzymatically, using a DNA polymerase or reverse transcriptase
which sequentially adds free nucleotides according to the
instructions of the template. The selection of a suitable enzyme to
achieve polymerization for a given template and primer is within
the purview of those skilled in the art. In certain embodiments,
the criteria for selection of polymerases includes lack exonuclease
activity or DNA polymerases which do not possess a strong
exonuclease activity. DNA polymerases with low exonuclease activity
for use in the present process may be isolated from natural sources
or produced through recombinant DNA techniques. Illustrative
examples of polymerases that may be used, are, without limitation,
T7 Sequenase v. 2.0, the Klenow Fragment of DNA polymerase I
lacking exonuclease activity, the Klenow Fragment of Taq
Polymerase, exo.- Pfu DNA polymerase, Vent. (exo.-) DNA polymerase,
and Deep Vent. (exo-) DNA polymerase.
In a particularly useful embodiment, the use of a boundary
oligonucleotide is avoided by removing unneeded portions of the
starting material by digestion. In this embodiment, which is shown
schematically in FIG. 2A, a restriction oligonucleotide 70 is
annealed to the starting material 100 at a preselected location.
The restriction oligonucleotide provides a double stranded portion
on the starting material containing a restriction site 72. Suitable
restriction sites, include, but are not limited to Xho I, Spe I,
Nhe1, Hind III, Nco I, Xma I, Bgl II, Bst I, and Pvu I. Upon
exposure to a suitable restriction enzyme, the starting material is
digested and thereby shortened to remove unnecessary sequence while
preserving the desired target sequence 12 (or portion thereof) to
be amplified on what will be used as the original template 110.
Once the original template 10 is obtained, a primer 20 is annealed
to the original template 110 (see FIG. 2B) adjacent to or
overlapping with the target sequence 12 as described above in
connection with previous embodiments. A strand of nucleic acid 40
complementary to the portion of the original template between the
3' end of the primer 20 and the 5' end of the original template 110
is polymerized. As those skilled in the art will appreciate, in
this embodiment where a restriction oligonucleotide is employed to
generate the original template, there is no need to use a boundary
oligonucleotide, because primer extension can be allowed to proceed
all the way to the 5' end of the shortened original template
110.
Once polymerization is complete (i.e., growing strand 40 reaches
the boundary oligonucleotide 30 or the 5' end of the shortened
original template 110), the newly synthesized complementary strand
is separated from the original template by any suitable denaturing
method including physical, chemical or enzymatic means. Strand
separation may also be induced by an enzyme from the class of
enzymes known as helicases or the enzyme RecA, which has helicase
activity and in the presence of riboATP is known to denature DNA.
The reaction conditions suitable for separating the strands of
nucleic acids with helicases are described by Cold Spring Harbor
Symposia on Quantitative Biology, Vol. XLIII "DNA: Replication and
Recombination" (New York: Cold Spring Harbor Laboratory, 1978), B.
Kuhn et al., "DNA Helicases", pp. 63-67, and techniques for using
RecA are reviewed in C. Radding, Ann. Rev. Genetics, 16:405-37
(1982).
The newly synthesized complementary strand thus includes sequences
provided by the primer 20 (e.g., the predetermined sequence 22, the
optional restriction site 23 and the annealing portion 25 of the
primer) as well as the newly synthesized portion 45 that is
complementary to the portion of the original template 10 between
the location at which the primer 20 was annealed to the original
template 10 and either the portion of the original template 10 to
which the boundary oligonucleotide 30 was annealed or through to
the shortened 5' end of the original template. See FIG. 3.
Optionally, multiple rounds of polymerization using the original
template and a primer are performed to produce multiple copies of
the newly synthesized complementary strand for use in subsequent
steps. It is contemplated that 2 to 10 rounds or more (preferably,
15-25 rounds) of linear amplification can be performed when a DNA
template is used. Making multiple copies of the newly synthesized
complementary strand at this point in the process (instead of
waiting until the entire engineered template is produced before
amplifying) helps ensure that accurate copies of the target
sequence are incorporated into the engineered templates ultimately
produced. It is believed that multiple rounds of polymerization
based on the original template provides a greater likelihood that a
better representation of all members of the library will be
achieved, therefore providing greater diversity compared to a
single round of polymerization.
The next step in preparing the engineered template involves
annealing a nested oligonucleotide 50 to the 3' end of the newly
synthesized complementary strand, for example as shown in FIG. 3.
As seen in FIG. 3, the nested oligonucleotide 50 provides a
template for further polymerization necessary to complete the
engineered template. Nested oligonucleotide 50 includes a portion
52 that does not hybridize and/or includes modified bases to the
newly synthesized complementary strand, thereby preventing the
nested oligonucleotide from serving as a primer. Nested
oligonucleotide 50 also includes a portion 55 that hybridizes to
the 3' end of the newly synthesized complementary strand. Nested
oligonucleotide 50 may optionally also define a restriction site
56. The final portion 58 of nested oligonucleotide 50 contains a
hairpin structure. From the point at which portion 55 extends
beyond the 3' end of the beginning the newly synthesized
complementary strand, the nested oligonucleotide serves as a
template for further polymerization to form the engineered
template. It should be understood that the nested oligo may contain
part of the target sequence (if part thereof was truncated in
forming the original template) or may include genes that encode a
polypeptide or protein (or portion thereof) such as, for example,
one or more CDR's or Framework regions or constant regions of an
antibody. It is also contemplated that a collection of nested
oligonucleotides having different sequences can be employed,
thereby providing a variety of templates which results in a library
of diverse products. Thus, polymerization will extend the newly
synthesized complementary strand by adding additional nucleic acid
60 that is complementary to the nested oligonucleotide as shown in
FIG. 3. Techniques for achieving polymerization are within the
purview of one skilled in the art. As previously noted, in
selecting a suitable polymerase, an enzyme lacking exonuclease
activity may be employed to prevent the 3' end of the nested oligo
from acting as a primer. Because of hairpin structure 50 of the
nested oligonucleotide, eventually the newly synthesized
complementary strand will turn back onto portion 45 of the same
strand which will then serve as the template for further
polymerization. Polymerization will continue until the end of the
primer is reached, at which point the newly synthesized strand will
terminate with a portion whose sequence is complementary to the
primer.
Once polymerization is complete, the engineered template 120 is
separated from the nested oligonucleotide 50 by techniques well
known to those skilled in the art such as, for example, heat
denaturation. The resulting engineered template 120 contains a
portion derived from the original primer 20, portion 45 that is
complementary to a portion of the original template, and portion 65
that is complementary to a portion of the nested oligonucleotide
and includes a hairpin structure 68, and a portion 69 that is
complementary to portion 45. (See FIGS. 4A and B.) The 3' end of
engineered template 120 includes a portion containing a sequence
that is complementary to primer 20. Thus, for example, as shown in
FIG. 4A, the 3' end of engineered template 120 includes portion 22'
containing a sequence that is complementary to the predetermined
sequence of portion 22 of primer 20. This allows for amplification
of the desired sequence contained within engineered template 120
using a single primer having the same sequence as the predetermined
sequence of primer portion 22 (or a primer that is complementary
thereto) using techniques known to those of ordinary skill in the
art.
As another example (shown in FIG. 4B), where mRNA is used as the
template and oligo dT is used as the primer, the 3' end of
engineered template 120 includes poly A portion that is
complementary to the oligo dT primer. In this case, any sequence
along portion 45 can be selected for use as the primer to be
annealed to portion 69 once the engineered template is denatured
for single primer amplification. Optionally, the primer may include
a non-annealing portion, such as, for example, a portion defining a
restriction site.
During single primer amplification, the presence of a polymerase
having exonuclease activity is preferred because such enzymes are
known to provide a "proofreading" function and have relatively
higher processivity compared to polymerases lacking exonuclease
activity.
Due to hairpin structure 68 there is internal self annealing
between the 5' end predetermined sequence and the 3' end sequence
which is complementary to the predetermined sequence on the
engineered template. Upon denaturation and addition of a primer
having the predetermined sequence, the primer will hybridize to the
template and amplification can proceed.
After amplification is performed, the products may be detected
using any of the techniques known to those skilled in the art.
Examples of methods used to detect nucleic acids include, without
limitation, hybridization with allele specific oligonucleotides,
restriction endonuclease cleavage, single-stranded conformational
polymorphism (SSCP), analysis.gel electrophoresis, ethidium bromide
staining, fluorescence resonance energy transfer, hairpin FRET
essay, and TaqMan assay.
Once the engineered nucleic acid is amplified a desired number of
times, restriction sites 23 and 66 or any internal restriction site
can be used to digest the strand so that the target nucleic acid
sequence can be ligated into a suitable expression vector. The
vector may then be used to transform an appropriate host organism
using standard methods to produce the polypeptide or protein
encoded by the target sequence.
In particularly useful embodiments, the methods described herein
are used to amplify target sequences encoding antibodies or
portions thereof, such as, for example the variable regions (either
light or heavy chain) using cDNA of an antibody. In this manner, a
library of antibodies can be amplified and screened. Thus, for
example, starting with a sample of antibody mRNA that is naturally
diverse, first strand cDNA can be produced and digested to provide
an original template. A primer can be designed to anneal upstream
to a selected complementary determining region (CDR) so that the
newly synthesized nucleic acid strand includes the CDR. By way of
example, if the target sequence is heavy chain CDR3, the primer may
be designed to anneal to the heavy chain framework one (FR1)
region. Those skilled in the art will readily envision how to
design appropriate primers to anneal to other upstream sites or to
reproduce other selected targets within the antibody cDNA based on
this disclosure.
The following Examples are provided to illustrate, but not limit,
the present invention(s):
Example 1
Amplification of a Repertoire of Ig Kappa Light Chain Variable
Genes
First Strand cDNA Synthesis
First strand cDNA to be used as the original template was generated
from 2 .mu.g of human peripheral blood lymphocyte (PBL) mRNA with
an oligo-dT primer using the SuperScript II First Strand Synthesis
Kit (Invitrogen) according to the manufacturer's instructions. The
1.sup.st strand cDNA product was purified over a QIAquick spin
column (QIAGEN PCR Purification Kit) and eluted in 400 .mu.L of
nuclease-free water.
Second Strand Linear Amplification (SSLA) in the Presence of
Blocking Oligonucleotide
The second strand cDNA reaction contained 5 .mu.L of 1.sup.st
strand cDNA original template, 0.5 .mu.M primer JMX26VK1a, 0.5
.mu.M blocking oligo CKLNA1, 0.2 mM dNTPs, 5 units of AmpliTaq Gold
DNA polymerase (Applied Biosystems), 1.times.GeneAmp Gold Buffer(15
mM Tris-HCl, pH 8.0, 50 mM KCl), and 1.5 mM MgCl.sub.2. The final
volume of the reaction was 98 .mu.L. The sequence of primer
JMX26VK1a, which hybridizes to the framework 1 region of VK1a
genes, was 5' GTC ACT CAC GAA CTC ACG ACT CAC GGA GAG CTC RAC ATC
CAG ATG ACC CAG 3' (SEQ ID NO: 1) where R is an equal mixture of A
and G. The sequence of the blocking oligo CKLNA1, which hybridizes
to the 5' end of the VK constant region, was 5' GAA CTG TGG CTG CAC
CAT CTG 3' (SEQ ID NO: 2), where the underlined bases are locked
nucleic acid (LNA) nucleotide analogues. After an initial heat
denaturation step of 94.degree. C. for 3 minutes, linear
amplification of 2.sup.nd strand cDNA was carried out for 20 cycles
of 94.degree. C. for 15 seconds, 56.degree. C. for 15 seconds, and
68.degree. C. for 1 minute.
Nested Oligo Extension Reaction
After the last cycle of linear amplification, 2 .mu.L of a
nested/hairpin oligo designated "JK14TSHP" was added to give a
final concentration of 20 .mu.M. The sequence of JK14TSHP was 5'
CCT TAG AGT CAC GCT AGC GAT TGA TTG ATT GAT TGATTG TTT GTG ACT CTA
AGG TTG GCG CGC CTT CGT TTG ATY TCC ACC TTG GTC C(ps)T(ps)G(ps)P 3'
(SEQ ID NO: 3) where Y is an equal mixture of C and T and (ps) are
phosphorothioate backbone linkages and P is a 3' propyl group. For
nested oligo extension reaction, two cycles of 94.degree. C. for 1
minute, 56.degree. C. for 15 seconds, and 72.degree. C. for 1
minute were performed, followed by a 10 minute incubation at
72.degree. C. to allow complete extension of the hairpin. The
reaction products were purified over a QIAquick spin column (QIAgen
PCR Purification Kit) and eluted in 50 .mu.L of nuclease-free
water.
Analysis of Engineered Template
The efficiency of the nested oligo extension reaction was
determined by amplifying the products with either a primer set
specific for the engineered product or a primer set that detects
all VK1a/JK14 second strand cDNA products (including the engineered
product). For specific detection of engineered product, a 10 .mu.L
aliquot was amplified for 20 or 25 cycles with primers designated
"JMX26" and "TSDP". Primer JMX26 hybridizes to the 5' end of
JMX26VK1a, the framework 1 primer used in the second strand cDNA
reaction. Primer TSDP hybridizes to the hairpin-loop sequence added
to the 3' ends of the second strand cDNAs in the nested oligo
extension reaction. The sequence of primer JMX26 was 5' GTC ACT CAC
GAA CTC ACG ACT CAC GG 3' (SEQ ID NO: 4). The sequence of primer
TSDP was 5' CAC GCT AGC GAT TGA TTG ATT G 3' (SEQ ID NO: 5). For
detection of all VK1a/JK14 second strand cDNA products a 10 .mu.L
aliquot was amplified for 20 or 25 cycles with primers JMX26 and
JK14. The sequence of primer JK 14, which hybridizes to the
framework 4 region of JK1 and JK4 genes, was 5' GAG GAG GAG GAG GAG
GAG GGC GCG CCT GAT YTC CAC CTT GGT CCC 3' (SEQ ID NO: 6). Both
reactions contained 1.times.GeneAmp Gold Buffer, 1.5 mM MgCl.sub.2,
7.5% glycerol, 0.2 mM dNTPs, and 0.5 .mu.M of each primer in a
final volume of 50 .mu.L.
The results with primers JMX26 and TSDP demonstrated the successful
production of nested oligo and extended VK stem-loop DNA when using
SSLA DNA that was blocked specifically with a boundary oligo.
Suitable controls showed that when using the nested oligo in the
presence of SSLA DNA that was not blocked, only a minimal amount of
amplified product was produced. Additional controls without the
nested oligo were negative. However, VK1a/JK14 second strand cDNA
products were detected equally among all tested samples.
Single Primer Amplification of the Stem-Loop cDNA Template
Conditions that were previously shown to amplify a 352 bp stem-15
bp loop DNA product were as follows: 10 pg of the stem-loop DNA, 2
.mu.M primer, 50 mM Tris-HCl, pH 9.0, 1.5 mM MgCl.sub.2, 15 mM
(NH.sub.4).sub.2SO.sub.4, 0.1% Triton X-100, 1.7 M betaine, 0.2 mM
dNTPs, and 2.5 units of Z-Taq DNA Polymerase (Takara Shuzo) in a
final volume of 50 .mu.L. The thermal cycling conditions were an
initial denaturation step of 96.degree. C. for 2.5 minutes, 35
cycles of 96.degree. C. for 30 seconds, 64.degree. C. for 30
seconds, 74.degree. C. for 1.5 minutes, and a final extension step
of 74.degree. C. for 10 minutes. Oligonucleotides containing the
modified bases 5-methyl-2'-deoxycytidine and/or
2-amino-2'-deoxyadenosine have been shown to prime much more
efficiently than unmodified oligonucleotides at primer binding
sites located within hairpin structures (Lebedev et al. 1996.
Genetic Analysis: Biomolecular Engineering 13, 15-21). These
modifications work by increasing the melting temperature of the
primer, allowing the annealing step of the amplification to be
performed at a higher temperature. JMX26 primers containing ten
5-methyl-2'-deoxycytidines or seven 2-amino-2'-deoxyadenosines have
been synthesized.
Cloning VK products
Amplified fragments are cloned by Sac I/Asc I into an appropriate
expression vector that contains, in frame, the remaining portion of
the kappa constant region. Suitable vectors include pRL5 and pRL4
vectors (described in U.S. Provisional Application 60/254,411, the
disclosure of which is incorporated herein by reference), fdtetDOG,
PHEN1, and pCANTAB5E. Individual kappa clones can be sequenced.
Expanding the Repertoire of VKappa Amplified Products
Further coverage of the VK repertoire is achieved by using the
above protocols with a panel of primers for the generation of the
second strand DNA. The primers contain JMX26 sequence, a Sac I
restriction site, and a region that anneals to 1.sup.st strand cDNA
in the framework 1 region of human antibody kappa light chain
genes. The antibody annealing sequences were derived from the VBase
database primers (www.mrc-cpe.cam.ac.uk/imt-doc/publicINTRO.html)
which were designed based on the known sequences of human
antibodies and are reported to cover the entire human antibody
repertoire of kappa light chain genes. Below is a list of suitable
primers:
JMX26Vk1a (SEQ ID NO:7)
GTCACTCACGAACTCACGACTCACGGAGAGCTCRACATCCAGATGACCCAG
JMX26Vk1b (SEQ ID NO: 8)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGMCATCCAGTTGACCCAG
JMX26Vk1C (SEQ ID NO: 9)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGCCATCCRGATGACCCAG
JMX26Vk1d (SEQ ID NO: 10)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGTCATCTGGATGACCCAG
JMX26Vk2a (SEQ ID NO: 11)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGATATTGTGATGACCCAG
JMX26Vk2b (SEQ ID NO: 12)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGATRTTGTGATGACTCAG
JMX26Vk3a (SEQ ID NO: 13)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTGTTGACRCAG
JMX26Vk3b (SEQ ID NO: 14)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATAGTGATGACGCAG
JMX26Vk3c (SEQ ID NO: 15)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTAATGACACAG
JMX26Vk4a (SEQ ID NO: 16)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGACATCGTGATGACCCAG
JMX26Vk5a (SEQ ID NO: 17)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAACGACACTCACGCAG
JMX26Vk6a (SEQ ID NO:18)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGAAATTGTGCTGACTCAG
JMX26 Vk6b (SEQ ID NO: 19)
GTCACTCACGAACTCACGACTCACGGAGAGCTCGATGTTGTGATGACACAG
In the foregoing sequences, R is an equal mixture of A and G, M is
an equal mixture of A and C, Y is an equal mixture of C and T, W is
an equal mixture of A and T, and S is an equal mixture of C and
G.
Example 2
Amplification of a Repertoire of IgM or IgG Heavy Chain or Lambda
Light Chain Variable Genes
Similar protocols are applied to the amplification of both heavy
chain and lambda light chain genes. JMX26, or another primer
without antibody specific sequences, is used for each of those
applications. If JMX26 is used, the second strand DNA is generated
with the primers listed below which contain JMX26 sequence, a
restriction site (Sac I for lambda, Xho I for heavy chains), and a
region that anneals to 1.sup.st strand cDNA in the framework 1
region of human antibody lambda light chain or heavy chain genes.
The antibody annealing sequences were derived from the VBase
database primers (www.mrc-cpe.cam.ac.uk/imt- doc/public/INTRO.html)
which were designed based on the known sequences of human
antibodies and are reported to cover the entire human antibody
repertoire of lambda light chain and heavy chain genes.
Lambda Light Chain Framework 1 Specific Primers:
JMX26VL1a (SEQ ID NO:20)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGTGCTGACTCAG
JMX26VL1b (SEQ ID NO: 21)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGTGYTGACGCAG
JMX264VL1C (SEQ ID NO: 22)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGTCGTGACGCAG
JMX26VL2 (SEQ ID NO: 23)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGTCTGCCCTGACTCAG
JMX26VL3a (SEQ ID NO:24)
GTCACTCACGAACTCACGACTCACGGAGAGCTCTCCTATGWGCTGACTCAG
JMX26VL3b (SEQ ID NO: 25)
GTCACTCACGAACTCACGACTCACGGAGAGCTCTCCTATGAGCTGACACAG
JMX26VL3c (SEQ ID NO: 26)
GTCACTCACGAACTCACGACTCACGGAGAGCTCTCTTCTGAGCTGACTCAG
JMX26VL3d (SEQ ID NO: 27)
GTCACTCACGAACTCACGACTCACGGAGAGCTCTCCTATGAGCTGATGCAG
JMX26VL4 (SEQ ID NO: 28)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGCYTGTGCTGACTCAA
JMX26VL5 (SEQ ID NO: 29)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGSCTGTGCTGACTCAG
JMX26VL6 (SEQ ID NO:30)
GTCACTCACGAACTCACGACTCACGGAGAGCTCAATTTTATGCTGACTCAG
JMX26VL7 (SEQ ID NO: 31)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGRCTGTGGTGACTCAG
JMX26VL8 (SEQ ID NO:32)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGACTGTGGTGACCCAG
JMX26VL4/9 (SEQ ID NO: 33)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCWGCCTGTGCTGACTCAG
JMX26VL10 (SEQ ID NO: 34)
GTCACTCACGAACTCACGACTCACGGAGAGCTCCAGGCAGGGCTGACTCAG
In the foregoing sequences (and throughout this disclosure), R is
an equal mixture of A and G, M is an equal mixture of A and C, Y is
an equal mixture of C and T, W is an equal mixture of A and T, and
S is an equal mixture of C and G.
Heavy Chain Framework 1 Specific Primers:
JMX24VH1a (SEQ ID NO: 35)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTKCAGCTGGTGCAG
JMX24VH1b (SEQ ID NO: 36)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTCCAGCTTGTGCAG
JMX26VH1c (SEQ ID NO: 37)
GTCACTCACGAACTCACGACTCACGGActcgagSAGGTCCAGCTGGTACAG
JMX26VH1d (SEQ ID NO: 38)
GTCACTCACGAACTCACGACTCACGGActcgagCARATGCAGCTGGTGCAG
JMX26VH2a (SEQ ID NO: 39)
GTCACTCACGAACTCACGACTCACGGActcgagCAGATCACCTTGAAGGAG
JMX26VH2b (SEQ ID NO: 40)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTCACCTTGARGGAG
JMX26VH3a (SEQ ID NO: 41)
GTCACTCACGAACTCACGACTCACGGActcgagGARGTGCAGCTGGTGGAG
JMX26VH3b (SEQ ID NO: 42)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTGCAGCTGGTGGAG
JMX26VH3c (SEQ ID NO: 43)
GTCACTCACGAACTCACGACTCACGGActcgagGAGGTGCAGCTGTTGGAG
JMX26VH4a (SEQ ID NO: 44)
GTCACTCACGAACTCACGACTCACGGActcgagCAGSTGCAGCTGCAGGAG
JMX26VH4b (SEQ ID NO: 45)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTGCAGCTACAGCAG
JMX26VH5a (SEQ ID NO: 46)
GTCACTCACGAACTCACGACTCACGGActcgagGARGTGCAGCTGGTGCAG
JMX26VH6a (SEQ ID NO: 47)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTACAGCTGCAGCAG
JMX26VH7a (SEQ ID NO: 48)
GTCACTCACGAACTCACGACTCACGGActcgagCAGGTSCAGCTGGTGCAA
In the foregoing sequences (and throughout this disclosure), R is
an equal mixture of A and G, K is an equal mixture of G and T, and
S is an equal mixture of C and G.
Blocking oligos for the constant domain of IgM, IgG, and lambda
chains are designed. Essentially, a region downstream of that
required for cloning the genes is identified, and within that
region, a sequence useful for annealing a blocking oligo is
determined. For example with IgG heavy chains, a native Apa I
restriction site present in the CH1 domain can be used for cloning.
Generally, the boundary oligo is located downstream of that native
restriction site. Compatible nested oligos are then designed which
contained all the elements described previously.
Once amplified, the lambda light chain genes are cloned as is
described above for the kappa light chain genes. Likewise,
amplified IgG heavy chain fragments are cloned by Xho I/Apa I into
an appropriate expression vector that contains, in frame, the
remaining portion of the CHI constant region. Suitable vectors
include pRL5, pRL4, fdtetDOG, PHEN1, and pCANTAB5E. Amplified IgM
heavy chain fragments are cloned by Xho I/EcoR I into an
appropriate expression vector that contains, in frame, the
remaining portion of the CH1 constant region. Like the Apa I
present natively in IgG genes, the EcoR I site is native to the IgM
CH1 domain. Libraries co-expressing both light chains and heavy
chains can be screened or selected for Fabs with the desired
binding activity.
Example 3
Amplification of a Repertoire of Human IgM Heavy Chain Genes
First Strand cDNA Synthesis
Human peripheral blood lymphocyte (PBL) mRNA was used as the
original template to generate the first strand cDNA with
ThermoScript RT-PCR System (Invitrogen Life Technologies). In
addition to oligo dT primer, a phosphoramidate oligonucleotide
(synthesized by Annovis Inc. Aston, PA) was also included in the
reverse transcription reaction. The phosphoramidate oligonucleotide
serves as a boundary for reverse transcriptase. The first strand
cDNA synthesis was terminated at the location where the
phosphoramidate oligonucleotide anneals with the mRNA. The
phosphoramidate oligonucleotide, PN-1, was designed to anneal with
the framework 1 region of immunoglobulin (Ig) heavy chain VH3 genes
and PN-VH5 was designed to anneal with the framework 1 region of
all the Ig heavy chain genes. A control for first strand cDNA
synthesis was also set up by not including the phosphoramidate
blocking oligonucleotide. The first strand cDNA product was
purified by QIAquick PCR Purification Kit (QIAGEN).
Phosphoramidate Framework 1 Blocking Oligonucleotides for Ig Heavy
Chain Genes have the Following Sequences: PN-1 5' GCCTCCCCCAGACTC
3' (SEQ ID NO:49) PN-VH5 5' GCTCCAGACTGCACCAGCTGCAC(C/T)TCGG 3'
(SEQ ID NO:50)
Examination of the Blocking Efficiency
The blocking efficiency in first strand cDNA synthesis was examined
by PCR reactions using blocking check primers and primer CM1,
dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction
buffer, and the first strand cDNA synthesis product. PCR was
performed on a PTC-200 thermal cycler (MJ Research) by heating to
94.degree. C. for 30 seconds and followed by cycles of 94.degree.
C. for 15 second, 60.degree. C. for 15 second, and 72.degree. C.
for one minute. The blocking check primers were designed to anneal
with the leader sequences of Ig heavy chain genes. The sequence of
CM1, which hybridizes with the CH1 region of IgM, was 5'
GCTCACACTAGTAGGCAGCTCAGCAATCAC 3' (SEQ ID NO: 51). Blocking was
analyzed by gel electrophoresis of the PCR products. With
appropriate number of cycles, less PCR product was observed from
the reverse transcription reactions containing the blocking
oligonucleotides than the one does not contain the blocking
oligonucleotides, an indication that termination of first strand
cDNA synthesis was provided by the hybridization of the blocking
oligonucleotides.
The sequences of the blocking check Primers for Ig heavy chain
genes have the following sequences: H1/7blck 5' C TGG ACC TGG AGG
ATC C 3' (SEQ ID NO:52) H1blck2 5' C TGG ACC TGG AGG GTC T 3' (SEQ
ID NO:53) H1blck3 5' C TGG ATT TGG AGG ATC C 3' (SEQ ID NO:54)
H2blck 5, GACACACTTTGCTCCACG 3' (SEQ ID NO:55) H2blck2 5' GAC ACA
CTT TGC TAC ACA 3' (SEQ ID NO:56) H3blck 5' TGGGGCTGAGCTGGGTTT 3'
(SEQ ID NO:57) H3blck2 5' TG GGA CTG AGC TGG ATT T 3' (SEQ ID
NO:58) H3blck3 5' TT GGG CTG AGC TGG ATT T 3' (SEQ ID NO:59)
H3blck4 5' TG GGG CTC CGC TGG GTT T 3' (SEQ ID NO:60) H3blck5 5' TT
GGG CTG AGC TGG CTT T 3' (SEQ ID NO:61) H3blck6 5' TT GGA CTG AGC
TGG GTT T 3' (SEQ ID NO:62) H3blck7 5' TT TGG CTG AGC TGG GTT T 3'
(SEQ ID NO:63) H4blck 5' AAACACCTGTGGTTCTTC 3' (SEQ ID NO:64)
H4blck2 5' AAG CAC CTG TGG TTT TTC 3' (SEQ ID NO:65) H5blck 5'
GGGTCAACCGCCATCCT 3' (SEQ ID NO:66) H6blck 5' TCTGTCTCCTTCCTCATC 3'
(SEQ ID NO:67) Second Strand cDNA Synthesis and Nesting
Oligonucleotide Extension Reaction:
The purified first strand cDNA synthesis product was used in a
nested oligo extension reaction with a hairpin-containing nesting
oligonucleotide, dNTPs, Advantage-2 DNA polymerase mix (Clontech),
and the reaction buffer. The extension reaction was performed with
a GeneAmp PCR System 9700 thermocyler (PE Applied Biosystems). It
was heated to 94.degree. C. for 30 seconds and followed by ten
cycles of 94.degree. C. for 15 seconds, appropriate annealing
temperature for each nesting oligonucleotide for 15 seconds,
ramping the temperature to 90.degree. C. at 10% of the normal
ramping rate, and 90.degree. C. for 30 seconds. The resulted heavy
chain gene should contain a hairpin structure.
Nesting Oligonucleotides for Ig VH1 Heavy Chain genes had the
following sequences:
hpVH1-1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAGGTGCAGCTGGTGCAG TCTGGGGCT
GAGGTGAAGAAGCCTG AAG 3' (SEQ ID NO: 68)
hpVH1-2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGCAGaTGCAGCTGGTGCAG
TCTGGGGCTGAGGTGAAGAAGaCTAAT 3' (SEQ ID NO: 69)
hpVH 1-3
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATG CAG CTG GTG CAG TCT
GGGCCT GAG GTG AAG AAG CCT ATT 3' (SEQ ID NO: 70)
hpVH1-4
5' CTCGAGGGCCCGCGAAAGCGGOCCCTCGAGGAGGTGCAGCTGGTGCAG
TCTGGGGCTGAGGTGAAGAAGCCTGAAG 3' (SEQ ID NO: 71)
Nesting Oligonucleotides for Ig VH2 Heavy Chain Genes:
hpVH2-1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATC ACC TTG AAG GAG TCT GGT
CCT ACG CTG GTG AAA CCC ACATAA 3' (SEQ ID NO: 72)
hpVH2-2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTC ACC TTG AAG GAG TCT GGT
CCT GYG CTG GTG AAA CCC AC TAA 3' Y:C/T (SEQ ID NO: 73)
Nesting Oligonucleotides for Ig VH3 Heavy Chain Genes:
hpVH3A1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG TCT GGG
GGA GGC TTG GT(C/A) CAG CCT GGGAAA 3' C/A: M(SEQ ID NO: 74)
hpVH3A2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGTCTGGG
GGAGGC(T/C)TGGT(A/C)AAGCCTGGGAAA 3' (SEQ ID NO: 75)
hpVH3A3
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT
CTGGGGGAGGTGTGGTACGGCCTGGGAAA 3' (SEQ ID NO: 76)
hpVH3A4
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGA
CTGGAGGAGGCTTGATCCAGCCTGGGAAG 3' (SEQ ID NO: 77)
hpVH3A5
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT
CTGGGGGAGTCGTGGTACAGCCTGGGAAA 3' (SEQ ID NO: 78)
hpVH3A6
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGGAGT CT
CGGGGAGTCTTGGTACAGCCTGGGAAA 3' (SEQ ID NO: 79)
hpVH3A7
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT GGG
GGA GGC TTG GTA CAG CCT GGCAAA 3' (SEQ ID NO: 80)
hpVH3A8
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT GGG
GGA GGC TTG GTC CAG CCT GGAAAA 3' (SEQ ID NO: 81)
hpVH3A9
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT GGG
GGA GGC TTA GTT CAG CCT GGGAAA 3' (SEQ ID NO: 82)
hpVH3A10
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GA G TCT GGG
GGA GGC TTG GTA CAG CCA GGGAAA 3' (SEQ ID NO: 83)
ots-hp-VH3b
5 CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGCAGGTGCAGCTGGTGGAGT
CTGGGGGAGGCGTGGTCCAGCCTGGGTTT 3' (SEQ ID NO: 84)
hp-VH3B2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGCAGGTGCAGCTGGTGGAGT
CTGGGGGAGGCTTGGTCAAGCCTGGAAAG 3' (SEQ ID NO: 85)
hpVH3C
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTGTTG GA G TCT GGG
GGA GGC TTG GTA CAG CCT GGGAAA 3' (SEQ ID NO: 86)
Nesting Oligonucleotides for Ig VH4 Heavy Chain Genes:
hpVH4-1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG STG CAG CTG CAG GA G TCG GGC
CCA GGA CTG GTG AAG CCT T AAA 3' S: C/G (SEQ ID NO: 87)
hpVH4-2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG CTG CAG CTG CAG GAG TCG GGC
TCA GGA CTG GTG AAG CCT T AAA 3' (SEQ ID NO: 88)
hpVH4-3
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG AG GTG CAG CTG CAGCAG TGG GGC GCA
GGA CTG TTG AAG CCT T AAT 3' (SEQ ID NO: 89)
Nesting Oligonucleotides for Ig VH5 Heavy Chain Genes:
othpVH52
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAGCTGGTGCAGT CT
GGAGCAGAGGTGAAAAAGCCCGGGGAAAA 3' (SEQ ID NO: 90)
Nesting Oligonucleotides for Ig VH6 Heavy Chain Genes:
hpVH6
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTA CAG CTG CAG CAG TCA GGT
CCA GGA CTG GTG AAG CCC AAA 3' (SEQ ID NO: 91)
Nesting Oligonucleotides for Ig VH7 Heavy Chain Genes:
hpVH7
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTG CAG CTG GTG CAA TCT GGG
TCT GAG TTG AAG AAG CCT ATA 3' (SEQ ID NO: 92)
Additional Ig Heavy Chain Nesting Oligonucleotides:
hpVH 3 kb1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCGACTGGTGGAG
TCTGGGGGAGACTTGGTAGAACCGGGGAAG 3' (SEQ ID NO: 93)
hpVH 3 kb2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGATGCAACTGGTGGAG
TCTGGGGGAGCCTTCGTCCAGCCGGGGAAG 3' (SEQ ID NO: 94)
Single Primer Amplification of IgM Hairpin-Containing Fd
Fragments
Products from the nesting oligo extension reaction (i.e. the
engineered template) were amplified using Advantage-2 DNA
polymerase mix (Clontech), the reaction buffer, dNTPs, and a single
primer named CM3 primer. The sequence for the CM3 primer, which
anneals with the CHI region of IgM, was: 5'
AGAATTTGACTAGTTGGCAAGAGGCACGTTCTTTTCTTTGTTGCCGT 3' (SEQ ID NO:
231). The amplification reaction was performed with a GeneAmp PCR
System 9700 thermocyler (PE Applied Biosystems). It was initially
heated to 94.degree. C. for 30 seconds and followed by thirty to
forty cycles of 94.degree. C. for 15 seconds, appropriate annealing
temperature for 15 seconds, ramping the temperature to 90.degree.
C. at 10% of the normal ramping speed, and at 90.degree. C. for 30
seconds. The amplified product was examined by electrophoresis to
be of the expected size, .about.0.7 kb. The amplified fragments
were cloned into an expression vector and their sequences were
confirmed to be human IgM.
Example 4
Amplification of a Repertoire of Human IgG Heavy Chain Genes from a
Donor Immunized with Hepatitis B Surface Antigen
First Strand cDNA Synthesis
The same protocol as example 3 is employed using mRNA of PBL from a
human donor immunized with hepatitis B surface antigen and the
phosphoramidate boundary oligonucleotides designed to anneal with
the leader sequence of the Ig heavy chain genes. The
phosphoramidate leader boundary oligonucleotides for Ig heavy chain
genes have the following sequences: PNVR3ld 5'CACCTCACACTGGACACCTTT
3' (SEQ ID NO:95) PNVH4ld 5'CTGGGACAGGACCCATCTGGG 3' (SEQ ID NO:96)
PNVH1ld 5'TGGGAGTGGGCACCTGTGG 3' (SEQ ID NO:97) PNVH2ld
5'CTGGGACAAGACCCATGAAG 3' (SEQ ID NO:98) PNVH5ld
5'TCGGAACAGACTCCTTGGAGA 3' (SEQ ID NO:99) PNVH6ld
5'CTGTGACAGGACACCCCATGG 3' (SEQ ID NO:100) Examination of the
Blocking Efficiency
The blocking efficiency in first strand cDNA synthesis is examined
by PCR reactions using dNTPs, Advantage-2 DNA polymerase mix
(Clontech), the reaction buffer, the first strand cDNA synthesis
product, the blocking check primers in Example 3, and the pooled
primer mixture of CG1Z, CG2speI, CG3speI, and CG4SpeI. The sequence
of primer CG1Z, which hybridized with the CH1 region of IgG1, is 5'
GCATGTACTAGTTTTGTCACAAGATTTGGG 3'. (SEQ ID NO: 101) The sequence of
primer CG2speI, which hybridized with the CH1 region of IgG2, is 5'
AAGGAAACTAGTTTTGCGCTCAACTGTCTTGTCCACCT 3'. (SEQ ID NO: 102) The
sequence of primer CG3speI, which hybridized with the CHI region of
IgG3, is 5' AAGGAAACTAGTGTCACCAAGTGGGGTTTTGAGCTC 3'. (SEQ ID NO:
103) The sequence of primer CG4speI, which hybridized with the CHI
region of IgG4, is 5' AAGGAAACTAGTACCATAGGACTCAACTCTCTTG 3'. (SEQ
ID NO: 104) PCR is performed on a PTC-200 thermal cycler (MJ
Research) by heating to 94.degree. C. for 30 seconds before the
following cycle is run, 94.degree. C. for 15 second, 60.degree. C.
for 15 second, and 72.degree. C. for one minute. The PCR products
were analyzed by gel electrophoresis. With appropriate number of
cycles less PCR products were observed from reverse transcription
reactions containing the blocking oligonucleotide than the one does
not contain blocking oligonucleotide, an indication that
termination of first strand cDNA synthesis was provided by
hybridization of the leader boundary oligonucleotides.
Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension
Reaction:
The same protocol as Example 3 is employed with nesting
oligonucleotides having the following sequences are used.
Nesting Oligonucleotides for Ig Heavy Chain VH3 Genes:
HpH3 L1
5'CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGSAGGTGCAGCTGGTGGAG TCYGAAA 3' where
S is an equal mixture of C and G, and Y is an equal mixture of T
and C (SEQ ID NO: 105)
HpH3L2
5'CTCGAGGGCCCGCGAAAGCGGGCCCTCGAGGAGGTGCAG CTG TTG GAG TCT GAAT 3'
(SEQ ID NO: 106)
HpH3L3
5'CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG ACT GATA
3' (SEQ ID NO: 107)
HpH3L4
5'CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG GAG TCT CAAA
3' (SEQ ID NO: 108)
Nesting Oligonucleotides for Ig Heavy Chain VH4 Genes:
HpH4L1
5'CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG STG CAG CTG CAG GAG TCG GAAA
3' where S is an equal mixture of C and G (SEQ ID NO: 109)
HpH4L2
5 CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG CTG CAG CTG CAG GAG TCC AAA 3'
(SEQ ID NO: 110)
HpH4L3
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTG CAG CTA CAG CAG TGG GAAA
3' (SEQ ID NO: 111)
Nesting Oligonucleotides for Ig Heavy Chain VH1 Genes:
HpH1L1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTB CAG CTK GTG CAG AAA 3'
where B is an equal mixture of C, G and T and K is an equal mixture
of G and T (SEQ ID NO: 112)
HpH1L2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG SAG GTC CAG CTG GTA CAG AAA 3'
where S is an equal mixture of C and G (SEQ ID NO: 113)
HpH1L3
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATG CAG CTG GTG CAG AAA 3'
(SEQ ID NO: 114)
HpH1L4
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAA ATG CAG CTG GTG CAG AAA 3'
(SEQ ID NO: 115)
Nesting Oligonucleotides for Ig Heavy Chain VH2 Genes:
HpH2L1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG ATC ACC TTG AAG GAG TCT AAA
3' (SEQ ID NO: 116)
HpH2L2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTC ACC TTG AAG GAG TCT AAA
3' (SEQ ID NO: 117)
Nesting Oligonucleotides for Ig Heavy Chain VH5 Genes:
HpH5L1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAG GTG CAG CTG GTG CAG AAA 3'
(SEQ ID NO: 118)
HpH5L2
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG GAA GTG CAG CTG GTG CAG AAA 3'
(SEQ ID NO: 119)
Nesting Oligonucleotides for Ig Heavy Chain VH6 Genes:
HpH6L1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTA CAG CTG CAG CAG TC AAA 3'
(SEQ ID NO: 120)
Nesting Oligonucleotides for Ig Heavy Chain VH7 Genes:
HpH7L1
5' CTCGAGGGCCCGCGAAAGCGGGCCCTCGAG CAG GTG CAG CTG GTG CAA TAAA 3'
(SEQ ID NO: 121)
Single Primer Amplification of Human IG Heavy Chain Fd Hairpin
Containing Fragments
The sample protocol as Example 3 was employed using CGIZ, CG2speI,
CG3speI, or CG4SpeI as the primer.
Cloning of Amplified IgG Heavy Chain Fd Fragments into a Phage
Display Vector
The amplified IgG heavy chain fd hairpin fragments are analyzed by
gel electrophoresis. The .about.0.7 kb fragment is separated from
the primers by cutting out the gel slice and the DNA was collected
by electroelution. The eluted DNA was precipitated by ethanol and
resuspended in water. It is digested with restriction enzymes XhoI
and SpeI and purified by the QIAquick PCR Purification Kit
(QIAGEN). The purified XhoI-SpeI fragment is ligated into a
suitable plasmid into which the light chain kappa genes amplified
from the same donor had previously been cloned. The ligated
reaction was transformed into E. coli XL-1 Blue strain
{F'proA.sup.+B.sup.+lac1.sub.q .DELTA. (lacZ) M15 Tn10/recA1 endA1
gyrA96thi-1 hsdR17 supE44 relA1 lac} by electroporation.
Selection of Human IgG Antibodies that Bind with the Hepatitis B
Surface Antigen
The XL-1 Blue cells electroporated with the ligation reaction of
the phagemid vector and the heavy chain Fd fragments were grown in
SOC medium at 37.degree. C. with shaking for one hour. SOC medium
is 20 mM glucose in SB medium which contains 1% MOPS hemisodium
salt, 3% Bacto Tryptone, and 2% Bacto Yeast Extract. Cells
transformed with the plasmid were selected by adding carbenicillin
to the culture and they were grown for two hours before infected
with a helper phage, VCSM13. After two hours XL-1 Blue cells
infected with the helper phage were selected by adding Kanamycin to
the culture and the infected cells were amplified overnight by
growing at 37.degree. C. with shaking. The next morning the
amplified phages were harvested by precipitating with polyethylene
glycol (PEG) from the culture supernatant. The PEG precipitated
phages were collected by centrifugation. They were resuspended in
1% bovine serum albumin (BSA) in TBS buffer and used in panning for
selecting human IgG antibodies that bind with the hepatitis B
surface antigen. The resuspended phages were bound with the
hepatitis B surface antigen immobilized on the ELISA plate
(Costar). The unbound phages were washed off with a washing buffer
(0.5% Tween 20 in PBS) and the bound phages were eluted off the
plate with a phage elution buffer (0.1M HCl/glycine, pH 2.2, 1
mg/ml BSA) and neutralized with a neutralization buffer (2M Tris
Base). The eluted phages were infected with E. coli ER strain {F'
proA.sub.+B.sup.+ lac1.sup.q .DELTA. (lacZ) M15/fhuA2 (ton
A).DELTA.(lac-proAB) supE thi-1 (hsdMS-mcrB) 5}, followed by
infection with VCSM13 helper phage. The panning procedure for
selecting antibodies bound to hepatitis B surface antigen were
repeated three more times. ELISA Screening of Antibody Clones that
Bind with the Hepatitis B Surface Antigen Phages eluted at the
fourth round of panning were infected with E. coli Top 10F' strain
{F' lac.sub.1.sup.q, Tn10 (Tet.sup.R mcrA .DELTA.(mrr-hsdRMS-mcrBC)
.PHI.8(lacZ .DELTA.m15 .DELTA.lacx74 deoR recA1 araD13
.DELTA.(ara-leu)7697 galU galK [sL(Str.sup.R) end A1 nupG) and
plated on LB-agar plates containing carbenicilin and tetracycline.
Individual clones were picked from the plates and grown overnight
in SB medium containing carbenicilin and tetracycline. The IgG Fab
fragment will be secreted into the culture supernatant. The next
morning cells were removed from these cultures by centrifugation
and the culture supernatant was screened in ELISA assay for binding
to hepatitis B surface antigen immobilized on the ELISA plates. To
reduce false positives the ELISA plates were pre-blocked with BSA
before binding with the Fab fragments in culture supernatant. The
non-binding Fab fragments were washed off by a washing solution
(0.05% Tween 20 in PBS). Following the wash, plates were incubated
with anti-human IgG (Fab').sub.2 conjugated with alkaline
phosphatase (Pierce) which reacts with p-Nitrophenyl phosphate
(Sigma), a chromogenic substrate that shows absorbance at OD405.
Positive binding clones were identified by a plate reader (Bio RAD
Model 1575) with light absorbance at OD405. Among the ninety-four
clones screened there were twenty-eight positive clones.
Characterization of the Hepatitis B Surface Antigen Binding Clones
The IgG heavy chain genes of positive clones from ELISA screening
were characterized by DNA sequencing. Plasmid DNA was extracted
from the positive clones and sequenced using primers leadVHpAX,
NdP, or SeqGZ (Retrogen, San Diego, Calif.). The sequencing primers
have the following sequences: VBVH3A 5'
GAGCCGCACGAGCCCCTCGAGGARGTGCAGCTGGTGGAG 3' (SEQ ID NO: 122) VBVH 3B
5' GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGGTGGAG 3' (SEQ ID NO: 123) VBVH
3C.sub.5' GAGCCGCACGAGCCCCTCGAGGAGGTGCAGCTGTTGGAG 3' (SEQ ID NO:
124) VBVH4A 5' GAGCCGCACGAGCCCCTCGAGCAG(CG)TGCAGCTGCAGGAG 3' (SEQ
ID NO: 125) VBVH4B 5' GAGCCGCACGAGCCCCTCGAGCAGGTGCAGCTACAGCAG 3'
(SEQ ID NO: 126) LeadVHPAX 5' GCGGCGCAGCCGGCGATGGCG 3' (SEQ ID NO:
127) NdP 5' AGCGTAGTCCGGAACGTCGTACGG (SEQ ID NO: 128) SeqGZ 5'
GAAGTAGTCCTTGACCAG 3' (SEQ ID NO: 129) The sequences of the
variable region of these IgG heavy chain genes from nineteen
positive clones are shown in FIG. 5. The great diversity of these
IgG heavy chain genes shows this method can efficiently amplify the
repertoire of human IgG heavy chain genes from immunized
donors.
Example 5
Amplification of a Repertoire of Human Light Chain Kappa Genes
First Strand cDNA Synthesis
The same protocol as example 3 is employed using the
phosphoramidate boundary oligonucleotides designed to hybridize
with the leader sequence of the kappa light chain genes. The
phosphoramidate leader boundary oligonucleotides for kappa light
chain genes have the following sequences:
PNK1Id: 5' T GTC ACA TCT GGC ACC TGG 3' (SEQ ID NO: 130)
PNK2Id: 5' TC CCC ACT GGA TCC AGG GAC 3' (SEQ ID NO: 131)
PNK3Id: 5.degree. C. TCC GOT GGT ATC TOG GAG 3' (SEQ ID NO:
132)
PNK4Id: 5' TC CCC GTA GGC ACC AGA GA 3' (SEQ ID NO: 133)
PNK5Id: 5' TC TGC CCT GGT AT C AGA GAT 3' (SEQ ID NO: 134)
PNK6Id: 5' ACC CCT GGA GGC TGG AAC 3' (SEQ ID NO: 135)
Examination of the Blocking Efficiency
The blocking efficiency in first Strand cDNA Synthesis was examined
by PCR reactions using blocking check primers and primer CK1DX2,
dNTPs, Advantage-2 DNA polymerase mix (Clontech), the reaction
buffer, and the first strand cDNA synthesis product. PCR was
performed on a PTC-200 thermal cycler (MJ Research) by heating to
94.degree. C. for 30 seconds and followed by cycles of 94.degree.
C. for 15 second, 60.degree. C. for 15 second, and 72.degree. C.
for one minute. The blocking check primers were designed to anneal
with the leader sequences of kappa light chain genes. The sequence
of CK1DX2, which hybridizes with the constant region of Kappa light
chain, was 5'
AGACAGTGAGCGCCGTCTAGAATTAACACTCTCCCCTGTTGAAGCTCTTTGTGAC
GGGCGAACTCAG 3'. (SEQ ID NO: 136) Blocking was analyzed by gel
electrophoresis of the PCR products. With appropriate number of
cycles less PCR products was observed from reverse transcription
reactions containing the blocking oligonucleotide than one that
does not contain blocking oligonucleotide, an indication that
termination of first strand cDNA synthesis was provided by
hybridization of the leader boundary oligonucleotides. Blocking
Check Primers for Kappa Light Chain Genes have the Following
Sequences: K1blck: 5'CTCCGAGGTGCCAGATGT 3' (SEQ ID NO: 137)
K1/2blck2: 5' GCT CAG CTC CTG GGG CT 3' (SEQ ID NO: 138) K2blck: 5'
GTCCCTGGATCCAGTGAG 3' (SEQ ID NO: 139) K3blck: 5'
CTCCCAGATACCACCGGA 3' (SEQ ID NO: 140) K3blck2: 5' GCG CAG CTT CTC
TTC CT 3' (SEQ ID NO: 141) K3blck3: 5' CAC AGC TTC TTC TTC CTC 3'
(SEQ ID NO: 142) K4blck: 5' ATCTCTGGTGCCTACGGG 3' (SEQ ID NO: 143)
K5blck: 5' ATCTCTGATACCAGGGCA 3' (SEQ ID NO: 144) K6blck: 5'
GTTCCAGCCTCCAGGGGT 3' (SEQ ID NO: 145) Second Strand cDNA Synthesis
and Nesting Oligonucleotide Extension Reaction: The same protocol
as Example 3 is employed using nesting oligonucleotides having the
following sequences: Nesting Oligonucleotides for Light Chain Kappa
Vk1: HpK1L1 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GMC ATC CAG ATG ACC
CAG TCT CCTAA 3' wherein M is an equal mixture of A and C (SEQ ID
NO: 146) HpK1L2 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAC ATC CAG ATG
ACC CAG TCT CC TAA 3' (SEQ ID NO: 147) HpK1L3 5'
GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GMC ATC CAG TTG ACC CAG TCT CC TAA
3' wherein M is an equal mixture of A and C (SEQ ID NO: 148) HpK1L4
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GCC ATC CGG ATG ACC CAG TCT CCTAT
3' (SEQ ID NO: 149) HpK1L5 5' GAGCTCGGCCCGCGAAAGCOGGCCGAGCTC GTC
ATC TGG ATG ACC CAG TCT CCTAT 3' (SEQ ID NO: 150) Nesting
Oligonucleotides for Light Chain Kappa Vk2: HpK2L1 5'
GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GTG ATG ACC CAG ACT CTTA 3'
(SEQ ID NO: 151) HpK2L2 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT GTT
GTG ATG ACT CAG TCT CCTAA 3' (SEQ ID NO: 152) HpK2L3 5'
GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAT ATT GTG ATG ACT CAG TCT CCTAA 3'
(SEQ ID NO: 153) Nesting Oligonucleotides for Light Chain Kappa
Vk3: HpK3L1 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT GTG TTG ACG
CAG TCT CCTAA 3' (SEQ ID NO: 154) HpK3L2 5'
GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATA GTG ATG ACG CAG TCT CCTAA3'
(SEQ ID NO: 155) HpK3L3 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT
GTA ATG ACA CAG TCT CCTAA3' (SEQ ID NO: 156) Nesting
Oligonucleotides for Light Chain Kappa Vk4: HpK4L1 5'
GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAC ATC GTG ATG ACC CAG TCT CCTAT3'
(SEQ ID NO: 157) Nesting Oligonucleotides for Light Chain Kappa
Vk5: HpK5L1 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ACG ACA CTC ACG
CAG TCT CCTAA3' (SEQ ID NO: 158) Nesting Oligonucleotides for Light
Chain Kappa Vk6: HpK6L1 5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC GAA ATT
GTG CTG ACT CAG TCT CCTAT3' (SEQ ID NO: 159) Single Primer
Amplification of Kappa Hairpin Fragments
The same protocol as Example 3 is employed using CK1DX2 as the
primer.
Example 6
Amplification of a Repertoire of Human Light Chain Lambda Genes
First Strand cDNA Synthesis
The same protocol as example 3 is employed using the following
phosphoramidate boundary oligonucleotides designed to hybridize
with the leader sequence of the lambda light chain genes. The
phosphoramidate boundary oligonucleotides for lambda light chain
genes have the sequences: PNL1Id: 5' CTG GGC CCA GGA CCC TGT GC 3'
(SEQ ID NO: 160) PNL2Id: 5' CTG GGC CCA GGA CCC TGT 3'. (SEQ ID NO:
161) PNL3Id: 5' GA GGC CAC AGA GCC TGT GCA GAG AGT GAG 3' (SEQ ID
NO: 162) PNL4Id1: 5' CAG AGC ACA GAG ACC TGT GGA3'(SEQ ID NO: 163)
PNL4Id2: 5' CTG GGA GAG AGA CCC TGT CCA3' (SEQ ID NO: 164) PNL5Id1:
5' CTG GGA GAG GGA ACC TGT GCA3' (SEQ ID NO: 165) PNL6Id1: 5' ATT
GGC CCA AGA ACC TGT GCA3' (SEQ ID NO: 166) PNL7Id1: 5' CTG AGA ATT
GGA CCC TGG GCA3' (SEQ ID NO: 167) PNL8Id1: 5' CTG AGA ATC CAC TCC
TGA TCC3' (SEQ ID NO: 168) PNL9Id1: 5' CTG GGA GAG GGA CCC TGT
GAG3' (SEQ ID NO: 169) PNL10Id1: 5' CTG GAC CAC TGA CAC TGC AGA3'
(SEQ ID NO: 170) Examination of the Blocking Efficiency
The same protocol as example 3 is employed using the following
blocking check primers and primer CL2DX2, dNTPs, Advantage-2 DNA
polymerase mix (Clontech), the reaction buffer, and the first
strand cDNA synthesis product. The blocking check primers have the
following sequences:
L1blck: 5' CAC TGY GCA GGG TCC TGG 3' (SEQ ID NO: 171)
L2blck: 5' CAG GGC ACA GGG TCC TGG 3' (SEQ ID NO: 172)
L3blck1: 5' TAC TGC ACA GGA TCC GTG 3' (SEQ ID NO: 173)
L3blck2: 5' CAC TTT ACA GGT TCT GTG 3' (SEQ ID NO: 174)
L3blck3: 5' TTC TGC ACA GTC TCT GAG 3' (SEQ ID NO: 175)
L3blck4: 5' CTC TGC ACA GGC TCT GAG 3' (SEQ ID NO: 176)
L3blck5: 5' CTT TGC TCA GGT TCT GTG 3' (SEQ ID NO: 177)
L3blck6: 5' CAC TGC ACA GGC TCT GTG 3' (SEQ ID NO: 178)
L3blck7: 5' CTC TAC ACA GGC TCT ATT 3' (SEQ ID NO: 179)
L3blck7: 5' CTC TGC ACA GTC TCT GTG 3' (SEQ ID NO: 180)
L4blck1: 5' TTC TCC ACA GGT CTC TGT 3' (SEQ ID NO: 181)
L4blck2: 5' CAC TGG ACA GGG TCT CTC 3' (SEQ ID NO: 182)
L5blck1: 5' CAC TGC ACA GGT TCC CTC 3' (SEQ ID NO: 183)
L6blck: 5' CAC TGC ACA GGT TCT TGG 3' (SEQ ID NO: 184)
L7blck: 5' TGC TGC CCA GGG TCC AAT 3' (SEQ ID NO: 185)
L8blck: 5' TAT GGA TCA GGA GTG GAT 3' (SEQ ID NO: 186)
L9blck: 5' CTC CTC ACA GGG TCC CTC 3' (SEQ ID NO: 187)
L10blck: 5' CAC TCT GCA GTG TCA GTG 3' (SEQ ID NO: 188)
The Sequence of CL2DX2, which hybridizes with the CL region of
Lambda genes, has this sequence: 5'
AGACAGTGACGCCGTCTAGAATTATGAACATTCTGTAGG 3' (SEQ ID NO: 189).
Second Strand cDNA Synthesis and Nesting Oligonucleotide Extension
Reaction:
The same protocol as Example 3 is employed using the nesting
oligonucleotides having the following sequences:
Nesting Oligonucleotides for Lambda Light Chain VL1:
HpL1.sub.L 1
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GTG CTG ACT CAG CCA CCAAA
3' (SEQ ID NO: 190)
HpL1L2
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GTG YTG ACG CAG CCG CCAAA
3' (SEQ ID NO: 191)
Nesting Oligonucleotides for Lambda Light Chain VL2:
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG TCT GCC CTG ACT CAG CCT
SAAA3' (SEQ ID NO: 192)
Nesting Oligonucleotides for Lambda Light Chain VL3:
HpL3L1
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACT CAG CCA
CYAAA3' (SEQ ID NO: 193)
HpL3L2
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACA CAG CYA CCAAT
3' (SEQ ID NO: 194)
HpL3L3
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC T CT TCT GAG CTG ACT CAG GAC
CCAAA 3' (SEQ ID NO: 195)
HpL3L4
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GTG CTG ACT CAG CCA CCAAA
3' (SEQ ID NO: 196)
HpL3L5
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ATG CAG CCA CCAAA
3' (SEQ ID NO: 197)
HpL3L6
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC TCC TAT GAG CTG ACA CAG CCA
TCAAA3' (SEQ ID NO: 198)
Nesting Oligonucleotides for Lambda Light Chain VL4:
HpL4L1
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CTG CCT GTG CTG ACT CAG CCC
CCAAA3' (SEQ ID NO: 199)
HpL4L2
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAA TCA
TCAAA3' (SEQ ID NO: 200)
HpL4L3
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CTT GTG CTG ACT CAA TCG
CCAAA3' (SEQ ID NO: 201)
Nesting Oligonucleotides for Lambda Light Chain VL5:
HpL5L1 5 e. 5b
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAG CCA
YCAAA3' (SEQ ID NO: 202)
HpL5L2 5c
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG GCT GTG CTG ACT CAG CCG
GCAAA3' (SEQ ID NO: 203)
Nesting Oligonucleotides for Lambda Light Chain VL6:
HpL6L1 6a
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC AAT TTT ATG CTG ACT CAG CCC
CAAAA3' (SEQ ID NO: 204)
Nesting Oligonucleotides for Lambda Light Chain VL7 and VL8:
HpL7/8L1
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG ACT GTG GTG ACY CAG GAG
CCAAA3' (SEQ ID NO: 205)
HpL7L2
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC G CAG GCT GTG GTG ACT CAG GAG
CCAAA3' (SEQ ID NO: 206)
Nesting Oligonucleotides for Lambda Light Chain VL9:
HpL9L
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG CCT GTG CTG ACT CAG CCA
CCAAA3' (SEQ ID NO: 207)
Nesting Oligonucleotides for Lambda Light Chain VL10:
5' GAGCTCGGCCCGCGAAAGCGGGCCGAGCTC CAG GCA GGG CTG ACT CAG CCA
CCAAA3' (SEQ ID NO: 208)
Single Primer Amplification of Lambda Hairpin Containing
Fragments
The same protocol as Example 3 is employed using CL2DX2 as the
primer.
Example 7
Amplification of a Repertoire of Human IgG Heavy Chain Genes from a
Donor Immunized with Hepatitis B Surface Antigen
First Strand cDNA Synthesis
The same protocol as example 3 was employed using mRNA of PBL from
a human donor immunized with hepatitis B surface antigen as the
original template using blocking oligonucleotides that anneal to
FR1 of the variable heavy chain.
Examination of the Blocking Efficiency
The same protocol as example 4 was employed.
Second Strand cDNA Synthesis And Nesting Oligonucleotide Extension
Reaction:
The same protocol as Example 3 was employed.
Single Primer Amplification of Human IgG Heavy Chain Fd Hairpin
Containing Fragments
The sample protocol as Example 4 was employed.
Cloning of Amplified IgG Heavy Chain Fd Fragments into a Phage
Display Vector
The sample protocol as Example 4 was employed.
Selection of Human IgG Antibodies that Bind with the Hepatitis B
Surface Antigen
The sample protocol as Example 4 was employed.
ELISA Screening of Antibody Clones that Bind with the Hepatitis B
Surface Antigen
The sample protocol as Example 4 was employed. Among the
ninety-four clones screened eighty clones are positive.
Characterization of the Hepatitis B Surface Antigen Binding
Clones
The sample protocol as Example 4 was employed. Sequences of the
variable regions of the heavy chain genes from fourteen positive
clones are listed in FIG. 6. The sequence diversity of these clones
and others produced shows this method can efficiently amplify the
repertoire of human heavy chain genes from immunized donors.
It will be understood that various modifications may be made to the
embodiments described herein. Therefore, the above description
should not be construed as limiting, but merely as exemplifications
of preferred embodiments. Those skilled in the art will envision
other modifications within the scope and spirit of this
disclosure.
SEQUENCE LISTINGS
1
231151DNAartificial sequenceprimer 1 gtcactcacg aactcacgac
tcacggagag ctcracatcc agatgaccca g 51 221DNAartificial
sequenceblocking oligonucleotide 2 gaactgtggc tgcaccatct g 21
393DNAartificial sequencenested/hairpin oligonucleoti de 3
ccttagagtc acgctagcga ttgattgatt gattgattgt ttgtgactct a aggttggcg
60 cgccttcgtt tgatytccac cttggtccnt ngn 93 426DNAartificial
sequenceprimer 4 gtcactcacg aactcacgac tcacgg 26 522DNAartificial
sequenceprimer 5 cacgctagcg attgattgat tg 22 645DNAartificial
sequenceprimer 6 gaggaggagg aggaggaggg cgcgcctgat ytccaccttg gtccc
45 751DNAartificial sequenceprimer 7 gtcactcacg aactcacgac
tcacggagag ctcracatcc agatgaccca g 51 851DNAartificial
sequenceprimer 8 gtcactcacg aactcacgac tcacggagag ctcgmcatcc
agttgaccca g 51 951DNAartificial sequenceprimer 9 gtcactcacg
aactcacgac tcacggagag ctcgccatcc rgatgaccca g 51 1051DNAartificial
sequenceprimer 10 gtcactcacg aactcacgac tcacggagag ctcgtcatct
ggatgaccca g 51 1151DNAartificial sequenceprimer 11 gtcactcacg
aactcacgac tcacggagag ctcgatattg tgatgaccca g 51 1251DNAartificial
sequenceprimer 12 gtcactcacg aactcacgac tcacggagag ctcgatrttg
tgatgactca g 51 1351DNAartificial sequenceprimer 13 gtcactcacg
aactcacgac tcacggagag ctcgaaattg tgttgacrca g 51 1451DNAartificial
sequenceprimer 14 gtcactcacg aactcacgac tcacggagag ctcgaaatag
tgatgacgca g 51 1551DNAartificial sequenceprimer 15 gtcactcacg
aactcacgac tcacggagag ctcgaaattg taatgacaca g 51 1651DNAartificial
sequenceprimer 16 gtcactcacg aactcacgac tcacggagag ctcgacatcg
tgatgaccca g 51 1751DNAartificial sequenceprimer 17 gtcactcacg
aactcacgac tcacggagag ctcgaaacga cactcacgca g 51 1851DNAartificial
sequenceprimer 18 gtcactcacg aactcacgac tcacggagag ctcgaaattg
tgctgactca g 51 1951DNAartificial sequenceprimer 19 gtcactcacg
aactcacgac tcacggagag ctcgatgttg tgatgacaca g 51 2051DNAartificial
sequenceprimer 20 gtcactcacg aactcacgac tcacggagag ctccagtctg
tgctgactca g 51 2151DNAartificial sequenceprimer 21 gtcactcacg
aactcacgac tcacggagag ctccagtctg tgytgacgca g 51 2251DNAartificial
sequenceprimer 22 gtcactcacg aactcacgac tcacggagag ctccagtctg
tcgtgacgca g 51 2351DNAartificial sequenceprimer 23 gtcactcacg
aactcacgac tcacggagag ctccagtctg ccctgactca g 51 2451DNAartificial
sequenceprimer 24 gtcactcacg aactcacgac tcacggagag ctctcctatg
wgctgactca g 51 2551DNAartificial sequenceprimer 25 gtcactcacg
aactcacgac tcacggagag ctctcctatg agctgacaca g 51 2651DNAartificial
sequenceprimer 26 gtcactcacg aactcacgac tcacggagag ctctcttctg
agctgactca g 51 2751DNAartificial sequenceprimer 27 gtcactcacg
aactcacgac tcacggagag ctctcctatg agctgatgca g 51 2851DNAartificial
sequenceprimer 28 gtcactcacg aactcacgac tcacggagag ctccagcytg
tgctgactca a 51 2951DNAartificial sequenceprimer 29 gtcactcacg
aactcacgac tcacggagag ctccagsctg tgctgactca g 51 3051DNAartificial
sequenceprimer 30 gtcactcacg aactcacgac tcacggagag ctcaatttta
tgctgactca g 51 3151DNAartificial sequenceprimer 31 gtcactcacg
aactcacgac tcacggagag ctccagrctg tggtgactca g 51 3251DNAartificial
sequenceprimer 32 gtcactcacg aactcacgac tcacggagag ctccagactg
tggtgaccca g 51 3351DNAartificial sequenceprimer 33 gtcactcacg
aactcacgac tcacggagag ctccwgcctg tgctgactca g 51 3451DNAartificial
sequenceprimer 34 gtcactcacg aactcacgac tcacggagag ctccaggcag
ggctgactca g 51 3551DNAartificial sequenceprimer 35 gtcactcacg
aactcacgac tcacggactc gagcaggtkc agctggtgca g 51 3651DNAartificial
sequenceprimer 36 gtcactcacg aactcacgac tcacggactc gagcaggtcc
agcttgtgca g 51 3751DNAartificial sequenceprimer 37 gtcactcacg
aactcacgac tcacggactc gagsaggtcc agctggtaca g 51 3851DNAartificial
sequenceprimer 38 gtcactcacg aactcacgac tcacggactc gagcaratgc
agctggtgca g 51 3951DNAartificial sequenceprimer 39 gtcactcacg
aactcacgac tcacggactc gagcagatca ccttgaagga g 51 4051DNAartificial
sequenceprimer 40 gtcactcacg aactcacgac tcacggactc gagcaggtca
ccttgargga g 51 4151DNAartificial sequenceprimer 41 gtcactcacg
aactcacgac tcacggactc gaggargtgc agctggtgga g 51 4251DNAartificial
sequenceprimer 42 gtcactcacg aactcacgac tcacggactc gagcaggtgc
agctggtgga g 51 4351DNAartificial sequenceprimer 43 gtcactcacg
aactcacgac tcacggactc gaggaggtgc agctgttgga g 51 4451DNAartificial
sequenceprimer 44 gtcactcacg aactcacgac tcacggactc gagcagstgc
agctgcagga g 51 4551DNAartificial sequenceprimer 45 gtcactcacg
aactcacgac tcacggactc gagcaggtgc agctacagca g 51 4651DNAartificial
sequenceprimer 46 gtcactcacg aactcacgac tcacggactc gaggargtgc
agctggtgca g 51 4751DNAartificial sequenceprimer 47 gtcactcacg
aactcacgac tcacggactc gagcaggtac agctgcagca g 51 4851DNAartificial
sequenceprimer 48 gtcactcacg aactcacgac tcacggactc gagcaggtsc
agctggtgca a 51 4915DNAartificial sequenceblocking oligonucleotide
49 gcctccccca gactc 15 5028DNAartificial sequenceblocking
oligonucleotide 50 gctccagact gcaccagctg cacntcgg 28
5130DNAartificial sequenceprimer 51 gctcacacta gtaggcagct
cagcaatcac 30 5217DNAartificial sequenceprimer 52 ctggacctgg
aggatcc 17 5317DNAartificial sequenceprimer 53 ctggacctgg agggtct
17 5417DNAartificial sequenceprimer 54 ctggatttgg aggatcc 17
5518DNAartificial sequenceprimer 55 gacacacttt gctccacg 18
5618DNAartificial sequenceprimer 56 gacacacttt gctacaca 18
5718DNAartificial sequenceprimer 57 tggggctgag ctgggttt 18
5818DNAartificial sequenceprimer 58 tgggactgag ctggattt 18
5918DNAartificial sequenceprimer 59 ttgggctgag ctggattt 18
6018DNAartificial sequenceprimer 60 tggggctccg ctgggttt 18
6118DNAartificial sequenceprimer 61 ttgggctgag ctggcttt 18
6218DNAartificial sequenceprimer 62 ttggactgag ctgggttt 18
6318DNAartificial sequenceprimer 63 tttggctgag ctgggttt 18
6418DNAartificial sequenceprimer 64 aaacacctgt ggttcttc 18
6518DNAartificial sequenceprimer 65 aagcacctgt ggtttttc 18
6617DNAartificial sequenceprimer 66 gggtcaaccg ccatcct 17
6718DNAartificial sequenceprimer 67 tctgtctcct tcctcatc 18
6876DNAartificial sequencenesting oligonucleotide 68 ctcgagggcc
cgcgaaagcg ggccctcgag caggtgcagc tggtgcagtc t ggggctgag 60
gtgaagaagc ctgaag 76 6975DNAartificial sequencenesting
oligonucleotide 69 ctcgagggcc cgcgaaagcg ggccctcgag cagatgcagc
tggtgcagtc t ggggctgag 60 gtgaagaaga ctaat 75 7075DNAartificial
sequencenesting oligonucleotide 70 ctcgagggcc cgcgaaagcg ggccctcgag
cagatgcagc tggtgcagtc t gggcctgag 60 gtgaagaagc ctatt 75
7176DNAartificial sequencenesting oligonucleotide 71 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tggtgcagtc t ggggctgag 60
gtgaagaagc ctgaag 76 7278DNAartificial sequencenesting
oligonucleotide 72 ctcgagggcc cgcgaaagcg ggccctcgag cagatcacct
tgaaggagtc t ggtcctacg 60 ctggtgaaac ccacataa 78 7377DNAartificial
sequencenesting oligonucleotide 73 ctcgagggcc cgcgaaagcg ggccctcgag
caggtcacct tgaaggagtc t ggtcctgyg 60 ctggtgaaac ccactaa 77
7478DNAartificial sequencenesting oligonucleotide 74 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc t gggggaggc 60
ttggtncagc ctgggaaa 78 7578DNAartificial sequencenesting
oligonucleotide 75 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtggagtc t gggggaggc 60 ntggtnaagc ctgggaaa 78 7678DNAartificial
sequencenesting oligonucleotide 76 ctcgagggcc cgcgaaagcg ggccctcgag
gaggtgcagc tggtggagtc t gggggaggt 60 gtggtacggc ctgggaaa 78
7778DNAartificial sequencenesting oligonucleotide 77 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tggtggagac t ggaggaggc 60
ttgatccagc ctgggaag 78 7878DNAartificial sequencenesting
oligonucleotide 78 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtggagtc t gggggagtc 60 gtggtacagc ctgggaaa 78 7978DNAartificial
sequencenesting oligonucleotide 79 ctcgagggcc cgcgaaagcg ggccctcgag
gaggtgcagc tggtggagtc t cggggagtc 60 ttggtacagc ctgggaaa 78
8078DNAartificial sequencenesting oligonucleotide 80 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc t gggggaggc 60
ttggtacagc ctggcaaa 78 8178DNAartificial sequencenesting
oligonucleotide 81 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtggagtc t gggggaggc 60 ttggtccagc ctggaaaa 78 8278DNAartificial
sequencenesting oligonucleotide 82 ctcgagggcc cgcgaaagcg ggccctcgag
gaggtgcagc tggtggagtc t gggggaggc 60 ttagttcagc ctgggaaa 78
8378DNAartificial sequencenesting oligonucleotide 83 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tggtggagtc t gggggaggc 60
ttggtacagc cagggaaa 78 8478DNAartificial sequencenesting
oligonucleotide 84 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc
tggtggagtc t gggggaggc 60 gtggtccagc ctgggttt 78 8578DNAartificial
sequencenesting oligonucleotide 85 ctcgagggcc cgcgaaagcg ggccctcgag
caggtgcagc tggtggagtc t gggggaggc 60 ttggtcaagc ctggaaag 78
8678DNAartificial sequencenesting oligonucleotide 86 ctcgagggcc
cgcgaaagcg ggccctcgag gaggtgcagc tgttggagtc t gggggaggc 60
ttggtacagc ctgggaaa 78 8776DNAartificial sequencenesting
oligonucleotide 87 ctcgagggcc cgcgaaagcg ggccctcgag cagstgcagc
tgcaggagtc g ggcccagga 60 ctggtgaagc cttaaa 76 8876DNAartificial
sequencenesting oligonucleotide 88 ctcgagggcc cgcgaaagcg ggccctcgag
cagctgcagc tgcaggagtc g ggctcagga 60 ctggtgaagc cttaaa 76
8975DNAartificial sequencenesting oligonucleotide 89 ctcgagggcc
cgcgaaagcg ggccctcgag aggtgcagct gcagcagtgg g gcgcaggac 60
tgttgaagcc ttaat 75 9080DNAartificial sequencenesting
oligonucleotide 90 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtgcagtc t ggagcagag 60 gtgaaaaagc ccggggaaaa 80
9175DNAartificial sequencenesting oligonucleotide 91 ctcgagggcc
cgcgaaagcg ggccctcgag caggtacagc tgcagcagtc a ggtccagga 60
ctggtgaagc ccaaa 75 9275DNAartificial sequencenesting
oligonucleotide 92 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc
tggtgcaatc t gggtctgag 60 ttgaagaagc ctata 75 9378DNAartificial
sequencenesting oligonucleotide 93 ctcgagggcc cgcgaaagcg ggccctcgag
gaggtgcgac tggtggagtc t gggggagac 60 ttggtagaac cggggaag 78
9478DNAartificial sequencenesting oligonucleotide 94 ctcgagggcc
cgcgaaagcg ggccctcgag gagatgcaac tggtggagtc t gggggagcc 60
ttcgtccagc cggggaag 78 9521DNAartificial sequenceboundary
oligonucleotide 95 cacctcacac tggacacctt t 21 9621DNAartificial
sequenceboundary oligonucleotide 96 ctgggacagg acccatctgg g 21
9719DNAartificial sequenceboundary oligonucleotide 97 tgggagtggg
cacctgtgg 19 9820DNAartificial sequenceboundary oligonucleotide 98
ctgggacaag acccatgaag 20 9921DNAartificial sequenceboundary
oligonucleotide 99 tcggaacaga ctccttggag a 21 10021DNAartificial
sequenceboundary oligonucleotide 100 ctgtgacagg acaccccatg g 21
10130DNAartificial sequenceprimer 101 gcatgtacta gttttgtcac
aagatttggg 30 10239DNAartificial sequenceprimer 102 aaggaaacta
gttttgcgct caactgtctt gtccacctt 39 10336DNAartificial
sequenceprimer 103 aaggaaacta gtgtcaccaa gtggggtttt gagctc 36
10437DNAartificial sequenceprimer 104 aaggaaacta gtaccatatt
tggactcaac tctcttg 37 10555DNAartificial sequencenesting
oligonucleotide 105 ctcgagggcc cgcgaaagcg ggccctcgag saggtgcagc
tggtggagtc y gaaa 55 10655DNAartificial sequencenesting
oligonucleotide 106 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tgttggagtc t gaat 55 10755DNAartificial sequencenesting
oligonucleotide 107 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtggagac t gata 55 10855DNAartificial sequencenesting
oligonucleotide 108 ctcgagggcc cgcgaaagcg ggccctcgag gaggtgcagc
tggtggagtc t caaa 55 10955DNAartificial sequencenesting
oligonucleotide 109 ctcgagggcc cgcgaaagcg ggccctcgag cagstgcagc
tgcaggagtc g gaaa 55 11054DNAartificial sequencenesting
oligonucleotide 110 ctcgagggcc cgcgaaagcg ggccctcgag cagctgcagc
tgcaggagtc c aaa 54 11155DNAartificial sequencenesting
oligonucleotide 111 ctcgagggcc cgcgaaagcg ggccctcgag caggtgcagc
tacagcagtg g gaaa 55 11251DNAartificial sequencenesting
oligonucleotide 112 ctcgagggcc cgcgaaagcg ggccctcgag caggtbcagc
tkgtgcagaa a 51 11351DNAartificial sequencenesting oligonucleotide
113 ctcgagggcc cgcgaaagcg ggccctcgag saggtccagc tggtacagaa a 51
11451DNAartificial sequencenesting oligonucleotide 114 ctcgagggcc
cgcgaaagcg ggccctcgag cagatgcagc tggtgcagaa a 51 11551DNAartificial
sequencenesting oligonucleotide 115 ctcgagggcc cgcgaaagcg
ggccctcgag caaatgcagc tggtgcagaa a 51 11654DNAartificial
sequencenesting oligonucleotide 116 ctcgagggcc cgcgaaagcg
ggccctcgag cagatcacct tgaaggagtc t aaa 54 11754DNAartificial
sequencenesting oligonucleotide 117 ctcgagggcc cgcgaaagcg
ggccctcgag caggtcacct tgaaggagtc t aaa 54 11851DNAartificial
sequencenesting oligonucleotide 118 ctcgagggcc cgcgaaagcg
ggccctcgag gaggtgcagc tggtgcagaa a 51 11951DNAartificial
sequencenesting oligonucleotide 119 ctcgagggcc cgcgaaagcg
ggccctcgag gaagtgcagc tggtgcagaa a 51 12053DNAartificial
sequencenesting oligonucleotide 120 ctcgagggcc cgcgaaagcg
ggccctcgag caggtacagc tgcagcagtc a aa 53 12152DNAartificial
sequencenesting oligonucleotide 121 ctcgagggcc cgcgaaagcg
ggccctcgag caggtgcagc tggtgcaata a a 52 12239DNAartificial
sequenceprimer 122 gagccgcacg agcccctcga ggargtgcag ctggtggag 39
12339DNAartificial sequenceprimer 123 gagccgcacg agcccctcga
ggaggtgcag ctggtggag 39 12439DNAartificial
sequenceprimer 124 gagccgcacg agcccctcga ggaggtgcag ctgttggag 39
12539DNAartificial sequenceprimer 125 gagccgcacg agcccctcga
gcagntgcag ctgcaggag 39 12639DNAartificial sequenceprimer 126
gagccgcacg agcccctcga gcaggtgcag ctacagcag 39 12721DNAartificial
sequenceprimer 127 gcggcgcagc cggcgatggc g 21 12824DNAartificial
sequenceprimer 128 agcgtagtcc ggaacgtcgt acgg 24 12918DNAartificial
sequenceprimer 129 gaagtagtcc ttgaccag 18 13019DNAartificial
sequenceboundary oligonucleotide 130 tgtcacatct ggcacctgg 19
13120DNAartificial sequenceboundary oligonucleotide 131 tccccactgg
atccagggac 20 13219DNAartificial sequenceboundary oligonucleotide
132 ctccggtggt atctgggag 19 13319DNAartificial sequenceboundary
oligonucleotide 133 tccccgtagg caccagaga 19 13420DNAartificial
sequenceboundary oligonucleotide 134 tctgccctgg tatcagagat 20
13519DNAartificial sequenceboundary oligonucleotide 135 cacccctgga
ggctggaac 19 13667DNAartificial sequenceprimer 136 agacagtgag
cgccgtctag aattaacact ctcccctgtt gaagctcttt g tgacgggcg 60 aactcag
67 13718DNAartificial sequenceprimer 137 ctccgaggtg ccagatgt 18
13817DNAartificial sequenceprimer 138 gctcagctcc tggggct 17
13918DNAartificial sequenceprimer 139 gtccctggat ccagtgag 18
14018DNAartificial sequenceprimer 140 ctcccagata ccaccgga 18
14117DNAartificial sequenceprimer 141 gcgcagcttc tcttcct 17
14218DNAartificial sequenceprimer 142 cacagcttct tcttcctc 18
14318DNAartificial sequenceprimer 143 atctctggtg cctacggg 18
14418DNAartificial sequenceprimer 144 atctctgata ccagggca 18
14518DNAartificial sequenceprimer 145 gttccagcct ccaggggt 18
14656DNAartificial sequencenesting oligonucleotide 146 gagctcggcc
cgcgaaagcg ggccgagctc gmcatccaga tgacccagtc t cctaa 56
14756DNAartificial sequencenesting oligonucleotide 147 gagctcggcc
cgcgaaagcg ggccgagctc aacatccaga tgacccagtc t cctaa 56
14856DNAartificial sequencenesting oligonucleotide 148 gagctcggcc
cgcgaaagcg ggccgagctc gmcatccagt tgacccagtc t cctaa 56
14956DNAartificial sequencenesting oligonucleotide 149 gagctcggcc
cgcgaaagcg ggccgagctc gccatccgga tgacccagtc t cctat 56
15056DNAartificial sequencenesting oligonucleotide 150 gagctcggcc
cgcgaaagcg ggccgagctc gtcatctgga tgacccagtc t cctat 56
15155DNAartificial sequencenesting oligonucleotide 151 gagctcggcc
cgcgaaagcg ggccgagctc gatattgtga tgacccagac t ctta 55
15256DNAartificial sequencenesting oligonucleotide 152 gagctcggcc
cgcgaaagcg ggccgagctc gatgttgtga tgactcagtc t cctaa 56
15356DNAartificial sequencenesting oligonucleotide 153 gagctcggcc
cgcgaaagcg ggccgagctc gatattgtga tgactcagtc t cctaa 56
15456DNAartificial sequencenesting oligonucleotide 154 gagctcggcc
cgcgaaagcg ggccgagctc gaaattgtgt tgacgcagtc t cctaa 56
15556DNAartificial sequencenesting oligonucleotide 155 gagctcggcc
cgcgaaagcg ggccgagctc gaaatagtga tgacgcagtc t cctaa 56
15656DNAartificial sequencenesting oligonucleotide 156 gagctcggcc
cgcgaaagcg ggccgagctc gaaattgtaa tgacacagtc t cctaa 56
15756DNAartificial sequencenesting oligonucleotide 157 gagctcggcc
cgcgaaagcg ggccgagctc gacatcgtga tgacccagtc t cctat 56
15856DNAartificial sequencenesting oligonucleotide 158 gagctcggcc
cgcgaaagcg ggccgagctc gaaacgacac tcacgcagtc t cctaa 56
15956DNAartificial sequencenesting oligonucleotide 159 gagctcggcc
cgcgaaagcg ggccgagctc gaaattgtgc tgactcagtc t cctat 56
16020DNAartificial sequenceboundary oligonucleotide 160 ctgggcccag
gaccctgtgc 20 16118DNAartificial sequenceboundary oligonucleotide
161 ctgggcccag gaccctgt 18 16229DNAartificial sequenceboundary
oligonucleotide 162 gaggccacag agcctgtgca gagagtgag 29
16321DNAartificial sequenceboundary oligonucleotide 163 cagagcacag
agacctgtgg a 21 16421DNAartificial sequenceboundary oligonucleotide
164 ctgggagaga gaccctgtcc a 21 16521DNAartificial sequenceboundary
oligonucleotide 165 ctgggagagg gaacctgtgc a 21 16621DNAartificial
sequenceboundary oligonucleotide 166 attggcccaa gaacctgtgc a 21
16721DNAartificial sequenceboundary oligonucleotide 167 ctgagaattg
gaccctgggc a 21 16821DNAartificial sequenceboundary oligonucleotide
168 ctgagaatcc actcctgatc c 21 16921DNAartificial sequenceboundary
oligonucleotide 169 ctgggagagg gaccctgtga g 21 17021DNAartificial
sequenceboundary oligonucleotide 170 ctggaccact gacactgcag a 21
17118DNAartificial sequenceprimer 171 cactgygcag ggtcctgg 18
17218DNAartificial sequenceprimer 172 cagggcacag ggtcctgg 18
17318DNAartificial sequenceprimer 173 tactgcacag gatccgtg 18
17418DNAartificial sequenceprimer 174 cactttacag gttctgtg 18
17518DNAartificial sequenceprimer 175 ttctgcacag tctctgag 18
17618DNAartificial sequenceprimer 176 ctctgcacag gctctgag 18
17718DNAartificial sequenceprimer 177 ctttgctcag gttctgtg 18
17818DNAartificial sequenceprimer 178 cactgcacag gctctgtg 18
17918DNAartificial sequenceprimer 179 ctctacacag gctctatt 18
18018DNAartificial sequenceprimer 180 ctctgcacag tctctgtg 18
18118DNAartificial sequenceprimer 181 ttctccacag gtctctgt 18
18218DNAartificial sequenceprimer 182 cactggacag ggtctctc 18
18318DNAartificial sequenceprimer 183 cactgcacag gttccctc 18
18418DNAartificial sequenceprimer 184 cactgcacag gttcttgg 18
18518DNAartificial sequenceprimer 185 tgctgcccag ggtccaat 18
18618DNAartificial sequenceprimer 186 tatggatcag gagtggat 18
18718DNAartificial sequenceprimer 187 ctcctcacag ggtccctc 18
18818DNAartificial sequenceprimer 188 cactctgcag tgtcagtg 18
18939DNAartificial sequenceprimer 189 agacagtgac gccgtctaga
attatgaaca ttctgtagg 39 19056DNAartificial sequencenesting
oligonucleotide 190 gagctcggcc cgcgaaagcg ggccgagctc cagtctgtgc
tgactcagcc a ccaaa 56 19156DNAartificial sequencenesting
oligonucleotide 191 gagctcggcc cgcgaaagcg ggccgagctc cagtctgtgy
tgacgcagcc g ccaaa 56 19255DNAartificial sequencenesting
oligonucleotide 192 gagctcggcc cgcgaaagcg ggccgagctc cagtctgccc
tgactcagcc t saaa 55 19356DNAartificial sequencenesting
oligonucleotide 193 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc
tgactcagcc a cyaaa 56 19456DNAartificial sequencenesting
oligonucleotide 194 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc
tgacacagcy a ccaat 56 19556DNAartificial sequencenesting
oligonucleotide 195 gagctcggcc cgcgaaagcg ggccgagctc tcttctgagc
tgactcagga c ccaaa 56 19656DNAartificial sequencenesting
oligonucleotide 196 gagctcggcc cgcgaaagcg ggccgagctc tcctatgtgc
tgactcagcc a ccaaa 56 19756DNAartificial sequencenesting
oligonucleotide 197 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc
tgatgcagcc a ccaaa 56 19856DNAartificial sequencenesting
oligonucleotide 198 gagctcggcc cgcgaaagcg ggccgagctc tcctatgagc
tgacacagcc a tcaaa 56 19956DNAartificial sequencenesting
oligonucleotide 199 gagctcggcc cgcgaaagcg ggccgagctc ctgcctgtgc
tgactcagcc c ccaaa 56 20056DNAartificial sequencenesting
oligonucleotide 200 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc
tgactcaatc a tcaaa 56 20156DNAartificial sequencenesting
oligonucleotide 201 gagctcggcc cgcgaaagcg ggccgagctc cagcttgtgc
tgactcaatc g ccaaa 56 20256DNAartificial sequencenesting
oligonucleotide 202 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc
tgactcagcc a ycaaa 56 20356DNAartificial sequencenesting
oligonucleotide 203 gagctcggcc cgcgaaagcg ggccgagctc caggctgtgc
tgactcagcc g gcaaa 56 20456DNAartificial sequencenesting
oligonucleotide 204 gagctcggcc cgcgaaagcg ggccgagctc aattttatgc
tgactcagcc c caaaa 56 20556DNAartificial sequencenesting
oligonucleotide 205 gagctcggcc cgcgaaagcg ggccgagctc cagactgtgg
tgacycagga g ccaaa 56 20657DNAartificial sequencenesting
oligonucleotide 206 gagctcggcc cgcgaaagcg ggccgagctc gcaggctgtg
gtgactcagg a gccaaa 57 20756DNAartificial sequencenesting
oligonucleotide 207 gagctcggcc cgcgaaagcg ggccgagctc cagcctgtgc
tgactcagcc a ccaaa 56 20856DNAartificial sequencenesting
oligonucleotide 208 gagctcggcc cgcgaaagcg ggccgagctc caggcagggc
tgactcagcc a ccaaa 56 209115PRTartificial sequencecloned antibody
209 Glu Ser Asp Gly Ala Val Val Gln Pro Gly G ly Ser Leu Arg Leu
Ser 1 5 10 15 Cys Ala Ala Ser Gly Phe Ile Phe Asp Asp P he Ala Met
His Trp Leu 20 25 30 Arg Gln Val Pro Gly Lys Gly Leu Gln Trp V al
Gly Leu Met Ser Trp 35 40 45 Asp Gly Val Ser Ala Tyr Tyr Ala Asp
Ser V al Glu Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Lys Lys
Asn Ala Leu T yr Leu Gln Met Asn Ser 65 70 75 80 Leu Gly Val Glu
Asp Thr Ala Leu Tyr Tyr C ys Ala Lys Asp Met Gly 85 90 95 Gly Gly
Leu Arg Phe Pro His Phe Trp Gly G ln Gly Thr Pro Val Thr 100 105
110 Val Ser Ala 115 210110PRTartificial sequencecloned antibody 210
Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys A la Ala Ser Gly Phe Thr 1
5 10 15 Leu Ser Ser Ser Ala Met Ser Trp Val Arg G ln Ala Pro Gly
Lys Gly 20 25 30 Leu Glu Phe Val Ala Val Ser Ser Gly Asn G ly Phe
Ser Thr Tyr Tyr 35 40 45 Gly Asp Ser Val Lys Gly Arg Phe Thr Ile S
er Arg Asp Asn Ser Lys 50 55 60 Asn Met Val Tyr Leu Gln Met Asp Ser
Leu A rg Ala Glu Asp Thr Ala 65 70 75 80 Lys Tyr His Cys Ala Lys
Val Arg Tyr Gly P ro Arg Ser His Phe Phe 85 90 95 Phe Asp Pro Trp
Gly Gln Gly Thr Leu Val T hr Val Ser Ser 100 105 110
211110PRTartificial sequencecloned antibody 211 Gln Pro Gly Gly Ser
Leu Arg Leu Ser Cys A la Ala Ser Gly Phe Thr 1 5 10 15 Leu Ser Ser
Ser Ala Met Ser Trp Val Arg G ln Ala Pro Gly Lys Gly 20 25 30 Leu
Glu Phe Val Ala Val Ser Ser Gly Asn G ly Phe Ser Thr Tyr Tyr 35 40
45 Gly Asp Ser Val Lys Gly Arg Phe Thr Ile S er Arg Asp Asn Ser Lys
50 55 60 Asn Met Val Tyr Leu Gln Met Asp Ser Leu A rg Ala Glu Asp
Thr Ala 65 70 75 80 Lys Tyr His Cys Ala Lys Val Arg Tyr Gly P ro
Arg Ser His Phe Phe 85 90 95 Phe Asp Pro Trp Gly Pro Gly Asn Pro
Gly H is Arg Leu Leu 100 105 110 212112PRTartificial sequencecloned
antibody 212 Ala Trp Tyr Ser Arg Gly Ser Pro Cys Leu S er Cys Ala
Ala Ser Gly 1 5 10 15 Phe Thr Leu Ser Ser Ser Ala Met Ser Trp V al
Arg Gln Ala Pro Gly 20 25 30 Lys Gly Leu Glu Phe Val Ala Val Ser
Ser G ly Asn Gly Phe Ser Thr 35 40 45 Tyr Tyr Gly Asp Ser Val Lys
Gly Arg Phe T hr Ile Ser Arg Asp Asn 50 55 60 Ser Lys Asn Met Val
Tyr Leu Gln Met Asp S er Leu Arg Ala Glu Asp 65 70 75 80 Thr Ala
Lys Tyr His Cys Ala Lys Val Arg T yr Gly Pro Arg Ser His 85 90 95
Phe Phe Phe Asp Pro Trp Gly Gln Gly Thr L eu Val Thr Val Ser Ser
100 105 110 213122PRTartificial sequencecloned antibody 213 Glu Ser
Asp Pro Gly Leu Val Lys Pro Ser G lu Thr Pro Ser Leu Thr 1 5 10 15
Cys Thr Val Ser Gly Gly Ser Ile Ser Ser T hr Met Tyr Phe Trp Gly 20
25 30 Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu G lu Trp Ile Ala Ser
Ile 35 40 45 Tyr Tyr Ser Gly Thr Thr Tyr Tyr Asn Pro S er Leu Arg
Ser Arg Val 50 55 60 Thr Met Ser Val Asp Thr Ser Lys Asn Gln L eu
Ser Leu Lys Leu Asn 65 70 75 80 Ser Val Thr Ala Ala Asp Thr Ala Val
Tyr T yr Cys Ala Arg Pro Thr 85 90 95 Ile Tyr Tyr Phe Asp Gly Arg
Thr Ser Tyr T yr Pro Gly Glu Ala Ala 100 105 110 Phe Asp Ile Trp
Gly Gln Gly Thr Thr Val 115 120 214121PRTartificial sequencecloned
antibody 214 Pro Gly Leu Val Lys Pro Ser Glu Thr Leu S er Leu Thr
Cys Thr Val 1 5 10 15 Ser Gly Gly Ser Ile Ser Asn Ile Met Tyr P he
Trp Gly Trp Ile Arg 20 25 30 Gln Pro Pro Gly Lys Gly Leu Glu Trp
Ile A la Ser Ile Tyr Tyr Ser 35 40 45 Gly Thr Thr Tyr Tyr Asn Pro
Ser Leu Arg S er Arg Val Thr Met Ser 50 55 60 Val Asp Thr Ser Lys
Asn Gln Leu Ser Leu L ys Leu Asn Ser Val Thr 65 70 75 80 Ala Ala
Asp Thr Ala Val Tyr Tyr Cys Ala A rg Pro Thr Ile Tyr Tyr 85 90 95
Phe Asp Gly Arg Thr Ser Tyr Tyr Pro Gly G lu Ala Ala Phe Asp Ile
100 105 110 Trp Gly Gln Gly Thr Thr Val Thr Val 115 120
215114PRTartificial sequencecloned antibody 215 Glu Ser Asp Pro Gly
Leu Val Gln Pro Ser G ln Thr Leu Ser Leu Thr 1 5 10 15 Cys Thr Val
Ser Gly Gly Ser Leu Arg Ser A sp Asp Tyr Tyr Trp Ser 20 25 30 Trp
Ile Arg Gln Ser Pro Gly Lys Gly Leu G lu Trp Ile Ala Tyr Ile 35 40
45 Ser Tyr Thr Gly Gly Thr Tyr Tyr Asn Pro S er Leu Lys Ser Arg Val
50 55 60 Thr Ile Ser Val Asp Thr Ser Arg Asn Gln P he Ser Leu Arg
Leu Arg 65 70 75 80 Ser Val Thr Ala Ala Asp Ser Ala Val Tyr P he
Cys Ala Ser Thr Thr 85 90 95 Ala Val Thr Thr Thr Phe Asp Tyr Trp
Gly A rg Gly Thr Leu Val Thr 100 105 110 Val Ser
216104PRTartificial sequencecloned antibody 216 Pro Val Gln Pro Leu
Glu Phe Thr Phe Thr A sp His Trp Met His Trp 1 5
10 15 Val Arg Gln Ala Pro Gly Lys Gly Leu Val T rp Leu Ala Arg Ile
Asn 20 25 30 Arg Asp Gly Ser Asp Thr Thr Tyr Ala Asp S er Val Thr
Gly Arg Phe 35 40 45 Thr Ile Ser Arg Asp Asn Gly Lys Asn Thr V al
Ser Leu Gln Met Asp 50 55 60 Ser Leu Ser Val Asp Asp Thr Ala Val
Tyr T yr Cys Ala Arg Gly Gly 65 70 75 80 His His Thr Val Leu Ser
Pro Leu Ser Asn T rp Phe Asp Pro Trp Gly 85 90 95 Gln Gly Thr Leu
Val Thr Val Ser 100 217110PRTartificial sequencecloned antibody 217
Glu Ser Glu Gly Gly Leu Val Gln Pro Gly G ly Ser Leu Arg Leu Ser 1
5 10 15 Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser T yr Ala Met Thr
Trp Val 20 25 30 Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp V al Ser
Thr Met Thr Gly 35 40 45 Ser Gly Gly Val Thr Tyr Tyr Ala Asp Val L
eu Lys Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn Ser Lys Asn Thr
Leu T yr Leu Gln Met Asn Ser 65 70 75 80 Leu Arg Ala Glu Asp Thr
Ala Val Tyr Tyr C ys Ala Lys Gly Tyr Gly 85 90 95 Leu Phe Asp Tyr
Trp Gly Gln Gly Thr Leu V al Thr Val Ser 100 105 110
218115PRTartificial sequencecloned antibody 218 Leu Ala Gly Val Glu
Val Val Gln Pro Gly G ly Ser Leu Arg Leu Ser 1 5 10 15 Cys Ala Ala
Ser Gly Phe Thr Phe Asp Asp T yr Ala Met His Trp Leu 20 25 30 Arg
Gln Ile Pro Gly Lys Gly Leu Gln Trp V al Ser Leu Leu Ser Trp 35 40
45 Asp Gly Val Ser Ala Tyr Tyr Ala Asp Ser V al Glu Gly Arg Phe Thr
50 55 60 Ile Ser Arg Asp Asn Lys Lys Asn Ser Leu T yr Leu Gln Met
Asn Ser 65 70 75 80 Leu Arg Ala Glu Asp Val Ala Leu Tyr Tyr C ys
Ala Lys Asp Met Gly 85 90 95 Gly Ala Gln Arg Leu Pro Asp His Trp
Gly G ln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
219114PRTartificial sequencecloned antibody 219 Gly Gly Gly Leu Val
Gln Pro Gly Ala Ser V al Lys Val Ser Cys Lys 1 5 10 15 Ala Ser Gly
Tyr Thr Phe Ser Asp Tyr Phe M et His Cys Val Arg Gln 20 25 30 Ala
Pro Gly Gln Gly Leu Glu Trp Met Gly L eu Val Asn Pro Thr Asn 35 40
45 Gly Tyr Thr Ala Tyr Ala Pro Lys Phe Gln G ly Arg Val Thr Met Thr
50 55 60 Arg Gln Arg Phe Thr Ser Thr Val Tyr Met G lu Leu Ser Ser
Leu Arg 65 70 75 80 Ser Glu Asp Thr Ala Val Tyr Phe Cys Ala A rg
Val Lys Ser Ser Asp 85 90 95 Ser Ile Asp Ala Phe Asp Ile Trp Gly
Gln G ly Thr Met Val Thr Val 100 105 110 Ser Ser
220103PRTartificial sequencecloned antibody 220 Arg Cys Pro Ala Lys
Leu Leu Asp Thr Pro P he Ser Val Tyr Phe Met 1 5 10 15 His Trp Val
Arg Gln Ala Pro Gly Gln Gly L eu Glu Trp Met Gly Leu 20 25 30 Val
Asn Pro Thr Asn Gly Tyr Thr Ala Tyr A la Pro Lys Phe Gln Gly 35 40
45 Arg Val Thr Met Thr Arg Gln Arg Phe Thr S er Thr Val Tyr Met Glu
50 55 60 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala V al Tyr Phe Cys
Ala Arg 65 70 75 80 Val Lys Ser Ser Asp Ser Ile Asp Ala Phe A sp
Ile Trp Gly Gln Gly 85 90 95 Thr Met Val Thr Val Ser Ser 100
221103PRTartificial sequencecloned antibody 221 Arg Cys Pro Ala Lys
Leu Leu Asp Thr Pro S er Gly Asp Tyr Phe Met 1 5 10 15 His Trp Val
Arg Gln Ala Pro Gly Gln Gly L eu Glu Trp Met Gly Leu 20 25 30 Val
Asn Pro Thr Asn Gly Tyr Thr Ala Tyr A la Pro Lys Phe Gln Gly 35 40
45 Arg Val Thr Met Thr Arg Gln Arg Phe Thr S er Thr Val Tyr Met Glu
50 55 60 Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala V al Tyr Phe Cys
Ala Arg 65 70 75 80 Val Lys Ser Ser Asp Ser Ile Asp Ala Phe A sp
Ile Trp Gly Gln Gly 85 90 95 Thr Met Val Thr Val Ser Ser 100
222115PRTartificial sequencecloned antibody 222 Ser Gly Gly Leu Val
Gln Arg Gly Ala Lys V al Leu Arg Leu Ser Cys 1 5 10 15 Val Ala Ser
Gly Phe Thr Phe Ser Ser Ser A la Met Ser Trp Val Arg 20 25 30 Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val S er Val Ile Ser Gly Asn 35 40
45 Gly Phe Ser Thr Tyr Tyr Ala Asp Ser Val L ys Arg Phe Thr Ile Ser
50 55 60 Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu G ln Met Asn Ser
Leu Arg 65 70 75 80 Ala Glu Asp Thr Ala Glu Tyr Tyr Cys Thr L ys
Val Lys Tyr Gly Ser 85 90 95 Gly Ser His Phe Trp Phe Asp Pro Trp
Gly G ln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115
22383PRTartificial sequencecloned antibody 223 Leu Gly Ser Pro Tyr
Ser Ser Ser Ala Met S er Trp Val Arg Gln Ala 1 5 10 15 Pro Gly Lys
Gly Leu Glu Xaa Val Ser Phe I le Ser Xaa Asn Gly Leu 20 25 30 Ser
Ala Tyr Tyr Ala Asp Ser Val Lys Gly A rg Phe Thr Ile Ser Arg 35 40
45 Asp Asn Ser Xaa Asn Thr Val Tyr Leu Gln M et Asn Ser Leu Arg Ser
50 55 60 Glu Asp Thr Ala Glu Tyr Tyr Cys Val Lys V al Xaa Tyr Gly
Ser Arg 65 70 75 80 Ser His Phe 224115PRTartificial sequencecloned
antibody 224 Val Glu Ser Gly Gly Val Val Gln Pro Gly A la Lys Val
Leu Arg Leu 1 5 10 15 Ser Cys Ala Ala Ser Gly Phe Ser Phe Glu A sp
Tyr Ala Met His Trp 20 25 30 Val Arg Gln Pro Pro Gly Lys Gly Leu
Glu T rp Val Ala Leu Ile Ser 35 40 45 Trp Asp Val Ile Ser Ala Tyr
Tyr Ala Asp S er Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Arg Asp
Asn Ser Lys Asn Ser L eu Tyr Leu Gln Met Asp 65 70 75 80 Ser Leu
Arg Pro Glu Asp Ser Gly Leu Tyr T yr Cys Gly Arg Asp Ile 85 90 95
Gly Gln Gln Arg Thr Met Asp Val Trp Gly G ln Gly Thr Thr Val Thr
100 105 110 Val Ser Ser 115 22598PRTartificial sequencecloned
antibody 225 Ala Ala Ser Gly Phe Ile Phe Asp Asp Phe A la Met His
Trp Phe Gln 1 5 10 15 Ala Val Pro Gly Lys Gly Leu Gln Trp Val G ly
Leu Met Ser Trp Asp 20 25 30 Gly Val Ser Ala Tyr Tyr Ala Asp Ser
Val G lu Gly Arg Phe Thr Ile 35 40 45 Ser Arg Asp Asn Lys Lys Asn
Ala Leu Tyr L eu Gln Met Asn Ser Leu 50 55 60 Gly Val Glu Asp Thr
Ala Leu Tyr Phe Cys A la Lys Asp Met Gly Gly 65 70 75 80 Gly Leu
Arg Phe Pro His Phe Trp Gly Gln G ly Thr Pro Val Thr Val 85 90 95
Ser Ala 226111PRTartificial sequencecloned antibody 226 Phe Trp Leu
Gly Gly Pro Trp Arg Leu Ser C ys Ala Val Ser Gly Tyr 1 5 10 15 Thr
Leu Ser Ser Ser Ala Met Ile Trp Val A rg Gln Pro Pro Gly Lys 20 25
30 Gly Leu Glu Phe Val Ser Val Ile Ser Gly A sn Gly Leu Ser Ala Tyr
35 40 45 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr I le Ser Arg Asp
Asn Ser 50 55 60 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser L eu Arg
Ala Glu Asp Thr 65 70 75 80 Ala Glu Tyr Tyr Cys Val Lys Val Lys Tyr
G ly Ser Arg Ser His Phe 85 90 95 Phe Phe Asp Ser Trp Gly Gln Gly
Thr Leu V al Ser Val Ser Pro 100 105 110 227115PRTartificial
sequencecloned antibody 227 Gly Gly Gly Leu Val Gln Pro Gly Ala Ser
L eu Arg Leu Ser Cys Val 1 5 10 15 Ala Ser Gly Phe Thr Leu Ser Ser
Ser Ala M et Ser Cys Val Arg Gln 20 25 30 Ala Pro Gly Lys Gly Leu
Glu Trp Val Ser V al Ser Ser Gly Asn Gly 35 40 45 Phe Ser Ala Tyr
Tyr Ala Asp Ser Val Lys G ly Arg Phe Thr Ile Ser 50 55 60 Arg Asp
Asn Ser Lys Asn Thr Leu Tyr Leu G ln Met Asn Ser Leu Val 65 70 75
80 Ala Glu Asp Thr Ala Glu Tyr Tyr Cys Thr L ys Val Asn Tyr Gly Ser
85 90 95 Arg Ser His Phe Tyr Phe Gly Ser Trp Gly H is Gly Thr Leu
Val Ile 100 105 110 Val Ser Ser 115 228114PRTartificial
sequencecloned antibody 228 Trp Gly Arg Arg Gly Pro Ala Trp Gly Val
P ro Val Gly Ser Pro Val 1 5 10 15 Gln Pro Leu Gly Tyr Thr Phe Asp
Asp Tyr A la Met His Trp Leu Arg 20 25 30 Gln Ile Pro Gly Lys Gly
Leu Gln Trp Val S er Leu Leu Ser Trp Asp 35 40 45 Gly Val Ser Ala
Tyr Tyr Ala Asp Ser Val G lu Gly Arg Phe Thr Ile 50 55 60 Ser Arg
Asp Asn Lys Lys Asn Ser Leu Tyr L eu Gln Met Asn Ser Leu 65 70 75
80 Val Ala Glu Asp Thr Ala Leu Tyr Phe Cys A la Lys Asp Met Gly Gly
85 90 95 Ala Gln Arg Leu Pro Asp His Trp Gly Gln G ly Thr Leu Val
Thr Val 100 105 110 Ser Ser 229115PRTartificial sequencecloned
antibody 229 Trp Thr Gly Gly Gly Val Val Gln Pro Gly G ly Ser Leu
Arg Val Ser 1 5 10 15 Val Ala Ala Ser Gly Tyr Thr Phe Asp Asp T yr
Ala Met His Trp Leu 20 25 30 Arg Gln Ile Pro Gly Lys Gly Leu Gln
Trp V al Ser Leu Leu Ser Trp 35 40 45 Asp Gly Val Ser Ala Tyr Tyr
Ala Asp Ser V al Glu Gly Arg Phe Thr 50 55 60 Ile Ser Arg Asp Asn
Xaa Lys Asn Ser Leu T yr Leu Gln Met Asn Ser 65 70 75 80 Leu Ile
Ala Glu Asp Thr Ala Leu Tyr Phe C ys Ala Lys Asp Met Gly 85 90 95
Gly Ala Gln Arg Leu Pro Asp His Trp Gly G ln Gly Thr Leu Val Thr
100 105 110 Val Ser Ser 115 230120PRTartificial sequencecloned
antibody 230 Ala Glu Ser Gly Gly Gly Val Val Gln Pro G ly Gly Ser
Leu Arg Leu 1 5 10 15 Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser A rg
Tyr Thr Leu Ser Trp 20 25 30 Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu T rp Val Ser Tyr Ile Ser 35 40 45 Thr Asp Gly Ser Thr Ile Tyr
Tyr Thr Asp S er Val Lys Gly Arg Phe 50 55 60 Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser L eu Ser Leu Gln Met Ile 65 70 75 80 Ser Leu
Arg Asp Glu Asp Thr Ala Val Tyr T yr Cys Ala Arg Val Phe 85 90 95
Phe Gly Gly Asn Phe Arg Ala His Trp Tyr P he Asp Leu Trp Gly Arg
100 105 110 Gly Thr Leu Val Ala Val Ser Ser 115 120
23147DNAartificial sequenceprimer 231 agaatttgac tagttggcaa
gaggcacgtt cttttctttg ttgccgt 47
* * * * *