U.S. patent number 5,580,730 [Application Number 08/293,638] was granted by the patent office on 1996-12-03 for enzyme digestion method for the detection of amplified dna.
This patent grant is currently assigned to Olympus America, Inc.. Invention is credited to Naoaki Okamoto.
United States Patent |
5,580,730 |
Okamoto |
December 3, 1996 |
**Please see images for:
( Certificate of Correction ) ** |
Enzyme digestion method for the detection of amplified DNA
Abstract
Qualitative and quantitative methods for detecting the presence
of double-stranded, non-5'-phosphorylated DNA in samples that may
also contain 5'-phosphorylated DNA and/or single-stranded DNA are
described. These methods involve treating the sample with an enzyme
that specifically degrades 5'-phosphorylated DNA together with an
enzyme that specifically degrades single-stranded DNA. More
specifically, methods are described for the detection of the
products of DNA amplification reactions, such as the polymerase
chain reaction (PCR), wherein post-amplification enzyme digestion
substantially reduces or eliminates background signals that are
apparently caused by the presence of template DNA and primers in
the sample after amplification is complete.
Inventors: |
Okamoto; Naoaki (South
Setauket, NY) |
Assignee: |
Olympus America, Inc. (Lake
Success, NY)
|
Family
ID: |
23129912 |
Appl.
No.: |
08/293,638 |
Filed: |
August 19, 1994 |
Current U.S.
Class: |
435/6.12; 435/15;
435/18; 435/91.1; 435/91.2; 536/24.33; 536/26.6 |
Current CPC
Class: |
C12Q
1/6848 (20130101); C12Q 1/6848 (20130101); C12Q
2563/107 (20130101); C12Q 2545/114 (20130101); C12Q
2521/325 (20130101) |
Current International
Class: |
C12Q
1/68 (20060101); C07H 021/04 (); C12P 019/34 ();
C12Q 001/68 () |
Field of
Search: |
;435/6,91.1,91.2,15,18
;536/26.6,24.33 ;935/77,78 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
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Fluorescence Detection Method for Human Leukocyte Antigen-DR Typing
Following Polymerase Chain Reaction Amplification with
Sequence-Specific Primer, Analytical Biochemistry 221,
340-347..
|
Primary Examiner: Jones; W. Gary
Assistant Examiner: Tran; Paul B.
Attorney, Agent or Firm: Kenyon & Kenyon
Claims
I claim:
1. A method for detecting the presence of double-stranded,
non-5'-phosphorylated DNA in a sample that may also contain
5'-phosphorylated DNA and single-stranded DNA, comprising:
(a) adding to the sample an enzyme that specifically degrades
single stranded DNA and an enzyme that specifically degrades DNA
having 5'-phosphorylated ends, and incubating under conditions that
permit the activity of said enzymes;
(b) detecting the presence of DNA in the sample of step (a) wherein
includes said DNA double stranded, non-5'-phosphorylated DNA.
2. A method for detecting the presence of one or more target DNA
sequences in a sample containing 5'-phosphorylated DNA,
comprising:
(a) carrying out enzymatic amplification of one or more portions of
the DNA in said sample to produce one or more amplification
products wherein at least a portion of each of said amplification
products is double stranded, non-5'-phosphorylated DNA;
(b) adding an enzyme that specifically degrades single stranded DNA
and an enzyme that specifically degrades DNA having
5'-phosphorylated ends, and incubating under conditions that permit
the activity of said enzymes;
(c) detecting the presence of DNA in the sample of step (b) wherein
said DNA includes double stranded, non-5'-phosphorylated DNA.
3. The method of claim 2 wherein said 5'-phosphorylated DNA is
biologically-derived.
4. The method of claim 2 wherein each of said steps are carried out
in succession by addition of reagents to the reaction mixture
resulting from the previous step, without any intervening
purifications or separations.
5. The method of claim 2 wherein all steps are carried out in the
same sample vessel.
6. The method of claim 5 wherein said sample vessel is a well of a
microtiter plate.
7. The method of claim 2 wherein said detection of DNA is
quantitative.
8. The method of claim 2 wherein said detection is qualitative, the
presence or absence of amplified DNA in an unknown sample being
determined by comparison with the results obtained using one or
more known samples.
9. The method of claim 2 wherein said step of detecting the
presence of DNA in the sample further comprises adding an
intercalating fluorophore to said sample and detecting the
fluorescence of the DNA when the resulting sample is excited by an
appropriate excitation beam.
10. The method of claim 9 wherein said intercalating fluorophore is
selected from the group of ethidium bromide (EtBr), ethidium
homodimer (EthD), Thiazole yellow dimer (YO-YO), Thiazole orange
dimer (TO-TO), and thiazole orange monomer (TO-PRO).
11. A method for detecting the presence of one or more target DNA
sequences in a sample containing 5'-phosphorylated DNA,
comprising:
(a) carrying out primed enzymatic transcription on one or more
portions of the DNA in said sample, using one or more primers that
do not have 5'-phosphorylated ends;
(b) adding to the DNA of said sample an enzyme that specifically
degrades single stranded DNA, and incubating under conditions that
permit the activity of said enzyme;
(c) adding to the DNA remaining after step (b) an enzyme that
specifically degrades DNA having 5'-phosphorylated ends, and
incubating under conditions that permit the activity of said
enzyme;
(d) detecting the presence of DNA in the sample of step (c) wherein
said DNA includes double stranded, non-5'-phosphorylated DNA.
12. A method for detecting the presence of one or more target DNA
sequences in a sample containing 5'-phosphorylated DNA,
comprising:
(a) amplifying one or more portions of the DNA in said sample by
polymerase chain reaction (PCR) to produce one or more
double-stranded, non-5'-phosphorylated amplification products;
(b) adding the enzymes lambda exonuclease and Exonuclease I and
incubating under conditions that permit the activity of said
enzymes;
(c) adding an intercalating fluorophore selected from the group
consisting of ethidium bromide (EtBr), ethidium homodimer (EthD),
Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and
thiazole orange monomer (TO-PRO);
(d) detecting the level of fluorescence emitted from the DNA in the
sample of step (c) when illuminated with an appropriate excitation
beam wherein said DNA includes double stranded,
non-5'-phosphorylated DNA.
13. The method of claim 12 wherein said level of fluorescence is
compared with the fluorescence levels observed when one or more
known samples were analyzed by the method of claim 13.
14. The method of claim 12 wherein said PCR amplification is
carried out using sequence specific primers.
15. The method of claim 14 wherein said sequence specific primers
are specific for one or more human leukocyte antigens (HLAs).
16. A homogeneous method for detecting the presence or absence of
one or more human leukocyte antigen (HLA) in a sample that is
substantially of human biological origin, comprising:
(a) using one or more HLA-sequence specific antigens in a
polymerase chain reaction (PCR) to amplify one or more portions of
the HLA-specific DNA in said sample;
(b) adding the enzymes lambda exonuclease and Exonuclease I and
incubating under conditions that permit the activity of said
enzymes;
(c) adding an intercalating fluorophore selected from the group of
ethidium bromide (EtBr), ethidium homodimer (EthD), Thiazole yellow
dimer (YO-YO), Thiazole orange dimer (TO-TO), and thiazole orange
monomer (TO-PRO);
(d) detecting the level of fluorescence emitted from the DNA in the
sample of step (c) when illuminated with an appropriate excitation
beam wherein said DNA includes double stranded,
non-5'-phosphorylated DNA.
Description
FIELD OF THE INVENTION
The present invention relates to qualitative and quantitative
methods for detecting the products of DNA amplification reactions,
such as the polymerase chain reaction (PCR). More specifically, the
present invention relates to the post-amplification use of an
enzyme that specifically degrades phosphorylated DNA together with
an enzyme that specifically degrades single-stranded DNA. Such
treatment serves to digest DNA other than that produced by
amplification, such that methods that detect DNA in the remaining
sample will yield qualitative and/or quantitative results that
substantially correlate with the presence and relative quantity of
amplified product present in said sample. More generally, the
present invention relates to the detection of double-stranded,
non-5'-phosphorylated DNA in samples that may also contain
5'-phosphorylated DNA and/or single-stranded DNA.
BACKGROUND OF THE INVENTION
It is often desirable to detect certain known or suspected target
sequences within samples of DNA that may be derived from biological
sources (either directly, or by indirect methods, e.g., reverse
transcription of DNA), or which may be derived by artificial means,
e.g., chemical synthesis or site-directed mutagenesis. Detection of
such target sequences may have utility in determining the presence
of infectious diseases such as HIV-I or Hepatitis B virus, as well
as in the detection of whether individuals carry genes for genetic
diseases such as sickle cell anemia or hemophilia. Such methods may
also be useful in identifying whether target sequences are
contained in populations of DNA produced by genetic manipulations
in vitro.
Perhaps the most powerful methods for detecting such target DNA
sequences take advantage of existing and emerging methods for
amplifying DNA sequences that may be present in samples in only
trace quantities. One particularly well-known amplification method
is the polymerase chain reaction (PCR). Once amplified, target DNA
sequences can be detected by a variety of methods for the detection
of specific DNA sequences, e.g., by gel electrophoresis or by
hybridization with labeled probes, or, if the amplified product is
present in substantially large quantities in relationship to the
DNA present in the original sample, by more simple methods that
detect the relative presence of DNA, e.g., by staining with
ethidium bromide, which becomes fluorescent upon intercalation
between nucleotide bases in double-stranded DNA.
Unfortunately, available methods for detecting DNA amplification
products have limitations that diminish the usefulness of such
methods for identifying target sequences. For example, gel
electrophoresis requires a considerable amount of sample handling,
and is thus not suitable for the rapid and cost-effective screening
of large numbers of samples. This handling can also lead to false
positives if even extremely small amounts of DNA are carried over
from sample to sample, as these may become subsequently
amplified.
A sense of the importance of such detection methods and the
limitations of those currently available can be readily appreciated
in the case of methods used to detect the presence of specific
HLA-class II molecules. These molecules are highly polymorphic
antigens which play a key role in the control of the immune
response. For example, the HLA-class II molecules DR and DQ are
involved in causing tissue rejection after tissue transplantation,
auto-immune diseases, and other immune-mediated disorders. There is
therefore a clinical need to be able to detect the presence of
these antigens in given individuals, in order to allow for tissue
type matching in anticipation of organ transplantation,
investigations into auto-immune and other HLA-related diseases, and
studies designed to explore the evolution and descendance of these
antigens.
Since the development of PCR (1,2), many amplification-dependant
approaches have been applied to HLA typing. For example,
restriction endonuclease digestion to produce PCR restriction
fragment length polymorphism (PCR-RFLP) has been used (3,4), and an
even more popular approach has been the hybridization of PCR
amplified products with sequence-specific oligonucleotide probes
(PCR-SSO) to distinguish between HLA alleles (5-7). Hybridization
and detection methods for PCR-SSO typing include the use of
non-radioactive labeled probes (8,9), microplate formats (10-12),
reverse dot blot formats (13,14) and automated large scale HLA
class 11 typing (15). A common drawback to these methods, however,
is the relatively long assay times needed--generally one to two
days--and their relatively high complexity and resulting high cost.
In addition, the necessity for sample transfers and washing steps
increases the chances that small amounts of amplified DNA might be
carried over between samples, creating the risk of false
positives.
Recently, a molecular typing method using sequence specific primer
amplification (PCR-SSP) has been described (16-18). This PCR-SSP
method is simple, useful and fast relative to PCR-SSO, since the
detection step is much simpler. In PCR-SSP, sequence specific
primers amplify only the complementary target allele, allowing
genetic variability to be detected with a high degree of
resolution. This method allows determination of HLA type simply by
whether or not amplification products (collectively called an
"amplicon") are present or absent following PCR.
In PCR-SSP, detection of the amplification products is usually done
by agarose gel electrophoresis followed by ethidium bromide (EtBr)
staining of the gel. Unfortunately, the electrophoresis process
takes a long time and is not very suitable for large number of
samples, which is a problem since each clinical sample requires
testing for many potential alleles. Gel electrophoresis also is not
easily adapted for automated HLA-DNA typing.
More recently, HLA-DNA PCR-SSP typing using Ethidium homodimer
(EthD) staining without electrophoresis has been described (19-20).
These methods still require the transfer of PCR products, and this
handling increases the chance that traces of amplified DNA will be
transferred from sample to sample, which can lead to contamination
and false positives.
In an effort to eliminate the need for sample transfers, a
homogeneous method for the detection of PCR amplified products
using Ethidium bromide (EtBr) fluorescence detection has been
developed (21,22). In this method, ethidium bromide is simply added
to the amplification reaction mixture; since the amplification
product should be present in a large amount relative to the DNA of
the starting sample, ethidium bromide fluorescence in a sample
where amplification occurred, i.e. where the target sequence was
present, is greater than the fluorescence in a sample where the
target sequence was lacking, and amplification did not occur.
Unfortunately, the template DNA, partial primer dimer, and primer
present in both positive and negative samples represents a
substantial background, making the discrimination between positive
and negative samples somewhat difficult and unreliable. The end
result is that the method has relatively low sensitivity and
reproducability. Prior to the present invention, no means for
reducing this background effect was known.
ADVANTAGES AND SUMMARY OF THE INVENTION
The present invention comprises new methods for the detection of
amplified DNA. More specifically, the present invention comprises
the use of post-amplification enzyme digestion of 5'-phosphorylated
DNA and single stranded DNA in order to degrade template DNA,
partial primer dimer, primer and other DNA. The amplified DNA is
not substantially 5'-phosphorylated or single stranded, and
therefore substantially escapes degradation. The end result is that
when presence of DNA in the remaining sample is then detected, the
signal obtained is substantially correlated with the presence of
amplified DNA in the sample.
One object of the present invention, then, is to provide a method
of detecting amplified DNA in which the background caused by the
presence of template DNA, partial primer-dimer, primer and other
DNA is substantially reduced.
It is a further object of the invention to provide a method for
detection of amplified DNA that has a higher degree of
reproducability and sensitivity than those methods available in the
prior art.
It is also an object of the invention to provide a method that
allows for the detection of amplified DNA in a homogeneous assay,
that is, an assay that can be carried out in a single vessel
without the need for transfer of any sample components.
As such, it is also an object of the invention to provide a method
of detecting amplified DNA in which the risks of sample
cross-contamination and resulting false positive results are
reduced.
It is an additional object of the invention to provide a method
that can allow for reliable, rapid analysis of multiple
samples.
It is also an object of the invention to provide a method of
detecting amplified DNA that is relatively simple, and likely to
result in a relatively low cost per analysis.
It is a further object of the present invention to provide a method
for the detection of amplified DNA that is amenable to
automation.
In one embodiment of the invention, a method for detecting the
presence of one or more target DNA sequences in a sample containing
5'-phosphorylated DNA is provided, which comprises the steps of (a)
carrying out enzymatic amplification of one or more portions of the
DNA in said sample to produce one or more substantially
double-stranded, substantially non-5'-phosphorylated amplification
products; (b) adding an enzyme that specifically degrades single
stranded DNA and an enzyme that specifically degrades DNA having
5'-phosphorylated ends, and incubating under conditions that permit
the activity of said enzymes; and (c) detecting the presence of DNA
in the sample.
In more detailed embodiments, the 5'-phosphorylated DNA present in
the sample may be biologically-derived, or alternatively may be
derived by genetic manipulation in vitro; the steps of the method
may be carried out in succession by addition of reagents to the
reaction mixture resulting from the previous step, without any
intervening purifications or separations; and all steps may be
carried out in the same sample vessel, which vessel may be a
microtiter plate. Detection of DNA may be quantitative or
qualitative, and may involve the use of intercalating fluorophores
such as ethidium bromide (EtBr), ethidium homodimer (EthD),
Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and
thiazole orange monomer (TO-PRO), or by other means. The method may
also be carried out by automated devices.
In another embodiment of the invention, a method is provided for
detecting the presence of one or more target DNA sequences in a
sample containing 5'-phosphorylated DNA, comprising the steps of
(a) carrying out primed enzymatic transcription on one or more
portions of the DNA in said sample, using one or more primers that
do not have 5'-phosphorylated ends; (b) adding to the DNA of said
sample an enzyme that specifically degrades single stranded DNA,
and incubating under conditions that permit the activity of said
enzyme; (c) adding to the DNA remaining after step (b) an enzyme
that specifically degrades DNA having 5'-phosphorylated ends, and
incubating under conditions that permit the activity of said
enzyme; and (d) detecting the presence of DNA in the sample.
Because the enzymatic digestion steps are carried out separately in
this embodiment, it is possible to use methods other than enzymatic
amplification, e.g., primer extension using a non-5'-phosphorylated
primer, which extended primer will be substantially converted to a
double-stranded, non-5'-phosphorylated DNA upon digestion with the
single strand-specific nuclease.
A further embodiment of the present invention provides a method for
detecting the presence of one or more target DNA sequences in a
sample containing 5'-phosphorylated DNA, comprising the steps of
(a) amplifying one or more portions of the DNA in said sample by
polymerase chain reaction (PCR) to produce one or more
double-stranded, non-phosphorylated amplification products; (b)
adding the enzymes lambda exonuclease and Exonuclease I and
incubating under conditions that permit the activity of said
enzymes; (c) adding an intercalating fluorophore selected from the
group of ethidium bromide (EtBr), ethidium homodimer (EthD),
Thiazole yellow dimer (YO-YO), Thiazole orange dimer (TO-TO), and
thiazole orange monomer (TO-PRO); and (d) detecting the level of
fluorescence emitted from the sample when illuminated with an
appropriate excitation beam.
More detailed embodiments of the invention comprise the above
method wherein said level of fluorescence is compared with the
fluorescence levels observed when one or more known samples were
analyzed by the same method; and/or wherein PCR amplification is
carried out using sequence specific primers, which may be specific
for one or more human leukocyte antigens (HLAs).
An additional embodiment of the present invention comprises a
homogeneous method for detecting the presence or absence of one or
more human leukocyte antigen (HLA) in a sample that is
substantially of human biological origin, comprising the steps of
(a) using one or more HLA-sequence specific antigens in a
polymerase chain reaction (PCR) to amplify one or more portions of
the HLA-specific DNA in said sample; (b) adding the enzymes lambda
exonuclease and Exonuclease I and incubating under conditions that
permit the activity of said enzymes; (c) adding an intercalating
fluorophore selected from the group of ethidium bromide (EtBr),
ethidium homodimer (EthD), Thiazole yellow dimer (YO-YO), Thiazole
orange dimer (TO-TO), and thiazole orange monomer (TO-PRO); and
detecting the level of fluorescence emitted from the sample when
illuminated with an appropriate excitation beam.
It is additionally an object of the present invention to provide
reagents kits that can be used to carry out the methods
described.
In one embodiment relating to this object of the invention, a kit
is provided for detecting the presence of one or more target DNA
sequences in a sample containing 5'-phosphorylated DNA, the kit
comprising one or more primers that do not have 5'-phosphorylated
ends; an enzyme that specifically degrades single stranded DNA; an
enzyme that specifically degrades DNA having 5'-phosphorylated
ends; and an intercalating fluorophore selected from the group of
ethidium bromide (EtBr), ethidium homodimer (EthD), Thiazole yellow
dimer (YO-YO), Thiazole orange dimer (TO-TO), and thiazole orange
monomer (TO-PRO).
Further embodiments of the kit of the present invention include
kits that further comprise reagent solutions having pH levels and
containing one or more reagent selected from the group of ions,
cofactors and metabolites such that, upon addition to the reaction
vessel, the composition of the resulting solution permits the
desired enzymatic reaction to occur; and/or further comprising a
thermostable DNA-dependent DNA polymerase, such as Taq polymerase.
The primers one such kits may be sequence-specific for one or more
portion of one or more human leucocyte antigen (HLA) DNA.
In addition to the foregoing, this invention is more generally
useful whenever it is desirable to detect the presence of
double-stranded, non-5'-phosphorylated DNA, where single-stranded
DNA and 5'-phosphorylated DNA present in that same sample might
cause an undesirable background signal.
The appended claims are hereby incorporated by reference as an
enumeration of the preferred embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a schematic illustration of how the enzymatic digestions
taught by the present invention provide for a reduction in the
presence of background DNAs;
FIGS. 2A-2D are a composite photograph of electrophoretic gels
showing that PCR-SSP analysis using HLA-DR sequence-specific
primers resulted in specific PCR-product banding patterns, as
expected; and
FIGS. 3A-3E show the results of a comparison of the fluorescence
obtained when PCR-SSP HLA typing was carried out using the
homogeneous method of the present invention and several different
fluorescent dyes at various concentrations, and comparing the
results obtained using positive and negative samples.
DETAILED DESCRIPTION OF THE INVENTION
The objects and advantages described above, and further objects and
advantages that will be apparent to those skilled in the art, stem
from a surprising discovery.
In my initial attempts to use PCR-SSP methods described by others
that involved the use of fluorescent intercalating dyes, I found
that there was an undesirably high signal observed even in samples
that did not contain the target sequence. This made it sometimes
difficult to distinguish between a positive and a negative result.
Although the prior art did not address this problem, I hypothesized
that this poor performance was due to high background fluorescence
from the template human DNA and unconsumed PCR primers.
Based on this hypothesis, I conceived that it might be possible to
use enzyme digestion to eliminate these sources of background
fluorescence, and that it might be possible to do so without the
need for intermediate purification of the DNA in the sample.
Although no single enzyme appeared to be able to achieve this, it
was decided that several enzymes with different specificities might
be able to act in concert to do so.
The first enzyme selected, Lambda exonuclease, is known to
selectively digest the phosphorylated strand of double stranded DNA
(27). Because biologically-derived DNA has at least one strand that
is substantially 5'-phosphorylated, it was hoped that digestion
with this enzyme would remove one strand of the template DNA, while
leaving the PCR amplified products substantially unaffected, as
they substantially lack the 5'phosphate. However, because of the
potentially great length of the human template DNA, it was unclear
whether this digestion would have any significant effect.
In a subsequent step, in hope of then digesting any resulting
single-stranded template DNA resulting from the first digestion and
also removing any unconsumed primer or other single-stranded
background DNA, the resulting sample was further digested by
Exonuclease 1. Exonuclease I is known to digest single-stranded
DNA, but not double-stranded DNA. Although single stranded DNA is
not generally expected to fluoresce in the presence of
intercalating dyes because those dyes only cause fluorescence of
double-stranded DNA, I had hypothesized that small regions of
secondary structure might cause a significant background if they
were able, singly or in combination, to form small regions of
hybridization. It was therefore hoped that this digestion would
provide a further diminution of the background.
As the examples appended hereto illustrate, the combined use of
these two enzymes gave a surprising degree of reduction in
background fluorescence, thus substantially increasing the
difference between the fluorescence of positive and negative
samples. Subsequent studies also demonstrated that it was possible
to combine these digestions into a single step, thus providing for
the first highly sensitive, highly reproducible homogeneous method
for detecting amplified DNA.
The overall scheme of the present invention is illustrated in FIG.
1. The left side of FIG. 1 illustrates that following PCR
amplification, positive samples (i.e., those containing the target
sequence) contain amplified DNA, and also contain the original
sample template DNA and unutilized primers. Negative samples,
(i.e., those not containing the target sequence) do not contain
amplified DNA, but do contain the template DNA and the added
primers. In prior art methods, fluorescent dye was added at this
point, and fluorescence was measured. Because of the presence of
template and primer DNA in both positive and negative samples,
substantial fluorescence occurred in both, and discerning the
increase in fluorescence in positive samples relative to negative
samples was problematic.
Referring again to FIG. 1, note that after the step labeled
"Selective Enzyme Digestion", the situation has considerably
changed. Now, the positive samples contain primarily the amplified
DNA, the template and primer having been substantially degraded.
Exonuclease I digestion may also have helped to digest partial
primer dimers that had single stranded regions. In negative
samples, there is no amplified DNA, and the template and the
primers have similarly been substantially degraded. When a
fluorescent dye is then added, the positive sample, containing
double-stranded amplified DNA, fluoresces strongly; whereas the
negative sample, lacking substantial quantities of DNA, fluoresces
weakly or not at all. When this result is compared to the results
obtained using prior art methods, the advantages of this reduction
in the background signal will be readily appreciated by those
skilled in the art.
The foregoing scheme is based upon the facts that PCR amplicons
have 5'-OH termini, since their 5' ends contain synthesized primer;
and that in contrast, human template DNA has 5'-phosphorylated
ends. Since Lambda exonuclease can digest only phosphorylated DNA,
it selectively digests the double stranded human DNA, presumably
down to single stranded DNA. Both the single stranded human DNA and
PCR primers are then degraded by Exonuclease 1. In contrast, PCR
amplified products, which are double stranded and have 5'-OH ends,
are not affected by these enzymes.
It is important to an understanding of the present invention to
note that all technical and scientific terms used herein, unless
otherwise defined, are intended to have the same meaning as
commonly understood by one of ordinary skill in the art; that
techniques employed herein are also those that are known to one of
ordinary skill in the art, unless stated otherwise; and that
publications mentioned herein are incorporated by reference.
It is also important to note that reference to particular buffers,
reagents and the like, or to some subclass of same, is not intended
to be limiting, but should be read to include all such related
materials that one of ordinary skill in the art would recognize as
being of interest or value in the particular context in which that
discussion is presented. For example, it is often possible to
substitute one buffer system for another, etc., such that a
different but known way is used to achieve the same goals as those
to which the use of a suggested method, material or composition is
directed.
It is additionally important to recognize that although Lambda
exonuclease and Exonuclease 1 are used in the examples provided
herein, the present invention is not limited to the use of those
particular enzymes. Any enzymes that have comparable activity may
be substituted, and such substitutions are within the scope of the
present invention. One source of such different enzymes may be to
derive enzymes that degrade 5'-phosphorylated DNA or
single-stranded DNA from different organisms than are the enzymes
used in the examples. Alternatively, enzymes structurally and
kinetically distinct from those mentioned may exist that have
similar activity. For example, the enzyme S1 nuclease also is known
to digest single-stranded DNA, and could be substituted for
Exonuclease 1.
Furthermore, even though human HLA antigen-specific primers are
used in the examples herein, the present invention is widely
applicable to testing for the presence of an enormous spectrum of
specific gene sequences. Moreover, it is not necessary that PCR-SSP
be the method used; the present invention is of utility anytime
amplification is carried out, and it is desirable to determine
whether the reaction has occurred or not, or to what degree it has
occurred. In addition, it is important to note that although PCR is
the amplification method used in the examples and preferred
embodiments described herein, the present invention is not
dependant thereon, and the use of the enzyme digestions described
herein in conjunction with other amplification methods, both those
known and not yet known, is well within the scope of the present
invention.
Finally, it is important to note that the present invention is not
limited to the use of all of the above-described discoveries or
embodiments together. Although combining them may indeed be
preferred, it is not necessary to the invention that all aspects be
used simultaneously.
It is necessary to a clear understanding of the present invention
to understand that a number of the terms used herein are not
intended to be limiting, even though common usage might suggest
otherwise. For example, where the term "transcription" is used,
this should be viewed broadly, e.g., to include the copying of one
strand of DNA to form a second strand, and also to include
iterative methods such as DNA amplification. The term "portions"
should similarly be viewed broadly, and would include the case
where a "portion" of a DNA strand is in fact the entire strand.
Where used herein, the term "sensitivity" is meant to refer to the
ability of an analytical method to detect small amounts of analyte.
Thus, as used here, a more sensitive method for the detection of
amplified DNA, for example, would be better able to detect small
amounts of such DNA than would a less sensitive method.
The term reproducibility as used herein refers to the general
ability of an analytical procedure to give the same result when
carried out repeatedly on aliquots of the same sample.
The term "target sequences" refers to sequences in a sample
template DNA, portions of which may be of particular interest.
Detection of the presence or absence of target sequences is, for
example, generally the object of the PCR-SSP method.
The term "amplicon" is used herein to mean a population of DNA
molecules that has been produced by amplification, e.g., by
PCR.
In describing the present invention, frequent mention is made to
detecting the presence of DNA in the enzyme-treated sample. It is
important to note that such detection can be carried out in a
variety of ways, all of which are within the scope of the present
invention. Such methods may detect specific sequences, detect
specific secondary structures, detect some other subset of the DNA
population, or simply detect total DNA. For example, the DNA
remaining after enzyme digestion can be detected by a dye that
intercalates between base pairs in double-stranded DNA, as
described in the examples that follow. Alternatively, it is also
possible, for example, to detect the presence of DNA by measuring
the absorbance of ultraviolet light by the sample, or by detecting
the binding of sequence-specific, fluorescently or radioactively
labeled DNA probes. These are offered as but a few examples, and
alternate means by which DNA can be detected will be readily
apparent to those skilled in the art.
In addition, it is also important to note that detection of the
presence of DNA may be qualitative or quantitative. The examples
describe homogeneous assays in which detection of DNA was
qualitative, i.e., where the samples were tested for the presence
or absence of amplified DNA products. However, many methods of
quantitative DNA analysis are known in the art, and such methods
can be used in the present invention to considerable advantage. For
example, by detecting the absolute or relative quantity of
amplified DNA in a sample, it should be possible to determine the
absolute or relative number of copies of a target sequence that
were in the original template DNA sample. This could be important,
for example, when it is desirable to determine whether one or both
of the alleles in the chromosomes of an individual carry a defect
associated with a genetic disease. Such an analysis could be used
to determine, for example, whether an as-yet unborn child will have
the phenotype associated with a recessive genetic defect, or to
determine what pattern of familial gene transmission can be
anticipated among an individual's offspring. These quantitative
approaches and their uses are well within the scope of the present
invention.
The use of the present invention is not limited to the treatment
and detection of amplified DNA, but extends to any situation
wherein it is desired to detect the presence of
non-5'-phosphorylated DNA in the presence of 5'-phosphorylated DNA
and single-stranded DNA. Many such situations can be envisioned.
For example, it is possible to add a non-5'-phosphorylated DNA
probe to a sample of heat-melted 5'-phosphorylated DNA, which will
result in the creation of double-stranded sequences that are
flanked on one or both ends by single-stranded sequences;
preferably, the 5'-phosphorylated end of the template will extend
beyond the probe and therefore be single-stranded. The sample could
then be digested with an enzyme that specifically degrades
single-stranded DNA, which would trim off the single stranded ends
and degrade any remaining primer. Next, the sample could be treated
with an enzyme that degrades 5'-phosphorylated DNA; this would
degrade any re-annealed template DNA, and if this latter digestion
is carried out in a manner that permits the continued activity of
the single stranded nuclease, this should degrade any
single-stranded sequences that result from the 5'-phosphorylated
DNA degrading enzyme. Detection of the DNA remaining after this
digestion will be indicative of the presence or absence of the
target sequence in the template DNA. This example is offered only
to illustrate that not only can amplified DNA can be detected by
the present methods, but DNA strands created by probe hybridization
and other methods can also be advantageously detected by the
present invention. Many other embodiments that do not involve DNA
amplification can also be envisioned by those skilled in the art,
and are within the scope of the present invention.
In carrying out the methods of the present invention, it is not
necessary that the two enzyme digestions be done simultaneously,
although this may be the most convenient approach in many cases,
but they can also be done separately. The digestions also need not
be done in any particular order if done separately, although using
one or the other first may have certain additional advantages, as
in the example of the paragraph above. In addition, it is not
necessary to halt the activity of one enzyme before adding the
other, as continued activity of the first added enzyme may not, in
a given experiment, be detrimental, and may actually be beneficial.
On the other hand, if desired, the activity of the first enzyme can
be halted before adding the second, for example, by heating the
sample to a temperature that denatures the first enzyme and then
cooling before adding the second enzyme. Many other ways of
inactivating the first enzyme can also be envisioned by those
skilled in the art. Of course, the second enzyme could also be
inactivated if desired, e.g., in a situation where a DNA probe will
subsequently be added.
The practice of the present invention does not require that
digestion by the enzymes described be carried to completion.
Although fairly complete digestion may in many instances be
preferred, one developing a specific application of the present
invention may find that more limited digestion provides a
sufficient reduction of the background, and that the increased
assay time or increased cost of a more complete digestion are not
justified by the incremental improvement in the assay that is
achieved.
Although the function of the present invention has here been
attributed to the digestion of a template 5'-phosphorylated DNA by
one enzyme and single-stranded probe and other single-stranded DNA
by another, it is important to note that the invention is not
limited in its application to situations where primers or probes
are added. It is possible that in some situations, it will
desirable to detect the presence of non-5'-phosphorylated DNA in a
sample containing 5'-phosphorylated DNA and no probe. In such an
instance, the single-stranded nuclease still is expected to be
important to use along with the 5'-phosphorylated DNA; as the
5'-phosphorylated DNA-degrading enzymes "chews" in from the ends of
one strand of a double strand, it likely leaves behind
single-stranded portions that can be degraded by the second
enzyme.
The 5'-phosphorylated DNA expected to serve as a template in
various embodiments of the present invention may be DNA of
biological origin, which is naturally 5'-phosphorylated. However,
it is also possible that 5'-phosphorylated DNA may be created by a
variety of synthetic means. For example, one might use
5'-phosphorylated probes to amplify a large region of a
non-5'-phosphorylated template DNA, and then use
non-5'-phosphorylated primers to amplify portions thereof. It is
also possible to enzymatically phosphorylate a given DNA sample. A
variety of other means by which the 5'-phosphorylated DNA in a
given sample might be originated can also be envisioned.
The detection of DNA after enzyme digestion is accomplished in the
following examples by the addition of an intercalating dye. In the
examples, the dye was added to the samples after enzyme digestion.
However, this is not meant to be limiting, as it is possible to add
the dye at any step, as will be readily apparent to those skilled
in the art.
In describing the present invention, some embodiments are described
as being "homogeneous" methods. This term refers to the potential
that the entire reaction can be carried out without the need for
intermediate purification or transfer of the sample or components
thereof.
In the examples, total DNA was first purified from biological
samples, and this was then used as an amplification template. This
should not be viewed as a limitation, as it may not be necessary in
a given circumstance to purify the DNA away from other biological
materials or other components in a starting sample.
Although definitions of some or all of the following abbreviations
may be set forth elsewhere herein, and although most if not all are
well recognized by those skilled in the art, their meaning are set
out hereinbelow for convenient reference.
PCR: Polymerase Chain Reaction
HLA: Human Leukocyte Antigen
SSP: Sequence Specific Primer
SSO: Sequence specific Oligonucleotide probe
RFLP: Restriction Fragment Length Polymorphism
EtBr: Ethidium Bromide
EthD: Ethidium homodimer
YO-YO: Thiazole yellow dimer
TO-TO: Thiazole orange dimer
TO-PRO: Thiazole orange monomer
The examples below describe in detail the use of a homogeneous
fluorescent detection method for HLA-SSP typing. Through this
combination of enzyme treatments, background fluorescence in PCR
reactions was decreased to about one third of that detected
untreated controls. The use of a new class of highly fluorescent
dimeric asymmetric cyanine fluorescence dyes that have a higher
affinity for double stranded DNA than EtBr and EthD (23,24)
augmented this result. Furthermore, this detection method is
homogeneous and does not require the transfer of PCR products
between microplates. PCR amplification, enzyme digestion, and
detection was carried out in the same well of the same plate
without transfer, and fluorescence was measured from that same well
using a microplate fluorescence reader, all within about 30
minutes. Elimination of sample transfer through the utilization of
microplate format PCR amplification and detection made possible the
faster, easier and more accurate method for detection of PCR
amplified sample. This approach is highly compatible with
automation and computerized data analysis. This progression towards
ease of use, high performance, and automation should make
homogeneous fluorescence detection methods especially suitable for
HLA typing laboratories and reference laboratories that deal with
large numbers of samples.
This method will be useful for the detection of PCR amplified
products not only for HLA typing, but also for infectious disease
testing of viral and bacterial DNA (e.g., HIV, HTLV, Herpes) and
for detection of mutations. Since blood borne virus detection
requires detection of a few target copies per .about.10.sup.5 human
cells (28), detection must be sensitive and specific. In earlier
homogeneous PCR detection methods, background fluorescence made it
difficult to detect a few copies of target DNA in large number of
cells (21). By using the improvements described herein, background
fluorescence can be greatly reduced and may be lowered sufficiently
to allow detection and quantification of even very low abundance
targets.
The described embodiments, examples and instructions are not
intended in any way to be limiting, as it should be readily
apparent to those skilled in the art how alternative means might be
used to achieve the results that this invention provides.
The following experimental methods, having been used by us to
demonstrate and illustrate the present invention, are described in
substantial detail hereinbelow. However, these details are not
intended to be limiting, and those skilled in the art will
appreciate that many other embodiments of the present invention are
possible.
Materials and Methods
DNA Extraction
DNA samples were extracted from peripheral blood leukocytes and
homozygous typing cell lines from the Tenth workshop panel (25).
Cells were lysed and digested by RNase and Protease K treatment and
their DNA extracts were purified by salting out and ethanol
precipitation (the protocol used was derived from a genomic DNA
isolation Kit obtained from the company Bio 101). Each aliquot of
extracted DNA was dissolved in H.sub.2 O at a concentration of 15
.mu.g/ml.
Preparation of sequence-Specific primers
The primers used were designed to obtain highly specific and
sensitive allele- and group-specific amplification for DR1 -DR1 8
as well as DR52 and DR53. Sequence specific primer pairs used for
DRB1, DRB3 and DRB4 typing are shown in Table 1. Primers were
TABLE 1
__________________________________________________________________________
Primer pairs for PCR-SSP HLA-DR typing HLA-DR Primer Pair Sequence
Serological name I.D. No. Sequence Amplified HLA-DR Alleles
Specificities
__________________________________________________________________________
DRBAMP-1 1 5'TTC TTG TGG CAG CTT AAG TTT3' DRB1*0101.0103 DR1 2
5'CCG CTG CAC TGT GAA GCT CT3' DRBAMP-2 3 5'CCT GTG GCA GCC TAA GAG
G3' DRB1*1501-1503 DR15(2) 4 5'CCG CTG CAC TGT GAA GCT CT3'
1601-1602 DR16(2) DRBAMP-15 5 5'CCT GTG GCA GCC TAA GAG G3'
DRB1*1501-1503 DR15(2) 6 5'TCC ACC GCG GCC CGC GC3' DRBAMP-16 7
5'CCT GTG GCA GCC TAA GAG G3' DRB1*1601-1602 DR16(2) 8 5'ACC GCG
GCG CGC CGC CTG TCT3' DRBAMP-3 9 5'CAC GTT TCT TGG AGT ACT CTA C3'
DRB1*0301-0303 DR17(3) 10 3'GCA GTA GTT GTC CAC CCG GC3' DR18(3)
DRBAMP-17 11 5'CAC GTT TCT TGG AGT ACT CTA C3'
DRB1*0301,1101-1104.sup.a DR17(3).DR11 12 5'AGC TCC GTC ACC GCC CGG
A3' 1201-1202.sup.a DR12 1301-1306(except 1303).sup.a DR13
DRBAMP-18 13 5'CAC GTT TCT TGG AGT ACT CTA C3' DRB1*0302-0303
DR18(3) 14 5'CTC CTG GTT ATG GAA GTA TCT C3' DRBAMP-4 15 5'GTT TCT
TGG AGC AGG TTA AA3' DRB1*0401-0412 DR4 16 5'CCG CTG CAC TGT GAA
GCT CT3' DRBAMP-11 17 5'CAC GTT TCT TGG AGT ACT CTA C3'
DRB1*1101-1104 DR11 18 5'CTG GCT GTT CCA GTA CTC CT3' DRBAMP-12 19
5'CAC GTT TCT TGG AGT ACT CTA C3' DRB1*1201-1202 DR12 20 5'GCT GTT
CCA GGA CTC GGC GA3' DRBAMP-7 21 5'CCT GTG GCA GGG TAA GTA TA3'
DRB1*0701-0702 DR7 22 5'CCC GTA GTT GTG TCT GCA CAC3' DRBAMP-8 23
5'GTA CTC TAC GGG TGA GTG TT3' DRB1*0801-0805 DR8 24 5'CTG CAG TAG
GTG TCC ACC AG3' DRBAMP-9 25 5'CGG AGC GGG TGC GGT AT3' DRB1*0901
DR9 26 5'CCC GTA GTT GTG TCT GCA CAC3' DRBAMP-10 27 5'CGG TTG CTG
GAA AGA CGC G3' DRB1*1001 DR10 28 5'CCG CTG CAC TGT GAA GCT CT3'
DRBAMP-13A 29 5'CAC GTT TCT TGG AGT ACT CTA C3' DRB1*1301,1302,1304
DR13 30 5'GTC CAC CGC GGC CCG CTC3' 1102-1103.sup.a DR11 DRBAMP-13B
31 5'CAC GTT TCT TGG AGT ACT CTA C3' DRB1*1303,1304 DR13 32 5'CTG
TTC CAG TAC TCG GCG CT3' 0801.sup.a,0803.sup.a,0805.sup.a DR8
DRBAMP-14A 33 5'CAC GTT TCT TGG AGT ACT CTA C3'
DRB1*1401,1404,1405,1407 DR14 34 5'CCA CCT CGG CCC GCC TCC3' 1408
DRBAMP-14B 35 5'CAC GTT TCT TGG AGT ACT CTA C3' DRB1*1402,1406,1409
DR14 36 5'CAC CGC GGC CCG CCT CTG3' DRBAMP-52 37 5'CCC CAG CAC GTT
TCT TGG AGC T3' DRB1*0101,0201-0202,0301 DR52 38 5'CCG CTG CAC TGT
GAA GCT CT3' DRBAMP-53 39 5'AGC GAG TGT GGA ACC TGA T3' DRB4*0101
DR53 40 5'CTC CAC AAC CCC GTA GTT GTA3'
__________________________________________________________________________
.sup.a Cross amplification
synthesized on a DNA synthesizer (Model 380 B, Applied Biosystem)
and purified by Oligonucleotide Purification Cartridges (Applied
Biosystems).
DNA Amplification
The PCR reaction mixture (25 .mu.l) consisted of 200 ng purified
sample DNA, PCR buffer (50 mM KCI, 2 mM MgCI2, 10 mM Tris-HCI, 0.1%
Triton X-100, pH 9.0), 200 .mu.M each dATP, dCTP, dGTP and dTTP,
0.5 .mu.M each sequence specific primer pair (0.2 .mu.M DRAMP-14A
primer pair), and 0.5 units Sequence grade Taq DNA polymerase
(Promega). Reagent, enzyme and sample DNA were placed in 96 well
polycarbonate microplates with plate covers (MJ Research) and
covered with 7 .mu.l of mineral oil to limit evaporative losses and
prevent contamination. PCR amplifications were carried out in a
PTC-100-96 V thermal cycler (MJ research). The temperature program
was: first denaturation step at 94.degree. C. for 2 min., followed
by 30 cycles of denaturation at 94.degree. C. for 30 s, then
annealing at 61.degree. C. for 50 s and extending at 72.degree. C.
for 30 s.
Electrophoresis
To confirm PCR amplification, PCR products were checked on 3%
Nusive agarose gels (FMC BioProducts) electrophoresed in
1.times.TBE buffer. 10 .mu.l of each PCR reaction was mixed with 5
.mu.l of dye glycerol mix (30% glycerol, 0.25% Bromphenol blue) and
loaded on the gels. Double-stranded DNA bands were visualized using
EtBr (0.5 g/ml).
Enzyme Treatment
After PCR amplification, 5 units of Lambda exonuclease (Pharmacia)
in 2 .mu.l PCR buffer and 10 units of Exonuclease I (United States
Biomedical) in 2 .mu.l PCR buffer were added to 15 .mu.l of each
PCR reaction mixture in the microplate well. The mixtures were
incubated at 37.degree. C. for 25 min and denatured at 70.degree.
C. for 5 min. in the thermal cycler.
Fluorescence detection
Fluorescent dyes Ethidium bromide, Ethidium homodimer, TO-PRO,
TO-TO and YO-YO were purchased from Molecular Probe. Each was
diluted and 1 .mu.l was added to each microwell after enzyme
treatment to yield final dye concentrations as described herein.
Fluorescence was detected using a Microplate fluorescence reader
(FLUOROSCAN 11, ICN Biomedicals). The polycarbonate microplate
wells were nested within the DYNATECH Micro FLUOR plate (U-bottom,
edge trimmed) to enhance signals and to prevent fluorescent signal
leaking between wells.
HLA DNA typing and serological typing of clinical samples
PCR amplification of PCR samples was done in polycarbonate
microplates (25 .mu.l reaction mix) with 20 sequence-specific
primers for HLA-DR typing. Ten microliters of PCR products were
analyzed by electrophoresis to confirm the amplification and 15
.mu.l of PCR products were detected by homogeneous fluorescence
detection following enzyme treatment. One microliter of 100 .mu.M
YO-YO was added to each well and the fluorescence was measured at
538 nm following excitation at 485 nm. Serological typings for the
HLA-DR locus were carried out by standard microlymphocytotoxicity
methods (26).
EXAMPLE 1
PCR-SSP amplification specificity
DNA samples from four clinical samples were subjected to 30 cycles
of PCR with the twenty sequence-specific primers of Table 1, which
were designed to detect the DRB1 alleles *0101-0103, 1501-3,
1601-2, 0301-0303, 0401-0412, 1101-1104, 1201-1202, 1301-1304,
1401-1402, 1404-1409, 0701-0702, 0801-0805, 0901, 1001, DRB3*0101,
0201-0202, 0301, and DRB4*0101. The HLA types of the four clinical
samples were: (A) DR7, DR12, (B) DR7, DR11, (C) DR1, DR9, and (D)
DR15, DR17. Each clinical specimen was tested with all 20 PCR
primers, and 10 .mu.l aliquots of each were run on agarose gels, as
shown in FIG. 2. The lanes of the gels in FIG. 2 are as follows: OX
174 Haelll-digested DNA molecular weight marker (lanes 1 and 12),
DRBAMP -1 (lane 2), DRBAMP -2 (lane 3), DRBAMP-15 (lane 4),
DRBAMP-16 (lane 5), DRBAMP-3 (lane 6), DRBAMP-17 (lane 7),
DRBAMP-18 (lane 8), DRBAMP-4 (lane 9), DRBAMP-11 (lane 10),
DRBAMP-12 (lane 11), DRBAMP-7 (lane 13), DRBAMP-8 (lane 14),
DRBAMP-9 (lane 15). DRBAMP-10 (lane 16), DRBAMP-13A (lane 17),
DRBAMP-13B (lane 18), DRBAMP-14A (lane 19), DRBAMP-14B (lane 20),
DRBAMP-52 (lane 21), and DRBAMP-53 (lane 22). These results clearly
show that specific amplification products were obtained in using
each primer.
Reduction of background fluorescence
Multiple aliquots of DNA extract from a DR 9-specific human blood
sample were amplified as described above using DRBAMP-1, DRBAMP-4,
DRBAMP-9, and DRBAMP-12 primers, and 15 .mu.l of each the resulting
reaction mixtures were (a) left untreated, (b) digested with Lambda
exonuclease, (c) digested with Exonuclease I, or (d) treated with
Lambda exonuclease and Exonuclease. Digestions were carried out
directly in the microtiter plate, and 1 .mu.l of TO-PRO was added
to each well. Fluorescence was then measured at 538 nm following
excitation at 485 nm.
The results are shown in Table 2. Through the combination of enzyme
treatments, background fluorescence in the PCR reactions,
apparently due to human DNA and primer, was decreased to about one
third of that observed in the untreated control samples. It appears
that
TABLE 2
__________________________________________________________________________
The effect of enzyme treatment on background fluorescence DR 9
amplification.sup.a Human DNA Primer PCR amplicon DRBAMP- 100 ng/15
.mu.l 0.4 .mu.M, 15 .mu.l HPLC purified 15 .mu.l 1 4 9 12
__________________________________________________________________________
No enzyme tratment 162.1.sup.b 80.6 670.1 161.5 170.6 644.5 166.1
Exonuclease I (10 U) 144.7 2.8 667.8 87.0 83.5 635.4 83.1 digestion
Lambda exonuclease 68.5 75.6 608.3 149.2 160.1 554.5 161.3
digestion (5 U) Exonuclease I (10 U) 43.8 2.2 615.0 71.5 63.9 557.1
60.2 Lambda exonuclease digestion (5 U)
__________________________________________________________________________
.sup.a PCR products from a complete PCRSSP amplification. (PCRSSP
of DR9 panel cell DNA with DRBAMP1, DRBAMP4, DRBAMP9 and DRBAMP12
primers) .sup.b After enzyme treatment, 1 .mu.l of 100 .mu.M TOPRO
was added to each sample. Fluorescence was measured at 538 nm
following excitation at 485 nm.
Exonuclease 1 digestion had the most dramatic effect, but Lambda
Exonuclease digestion also had a measurable effect; the combination
of the two was clearly preferable.
EXAMPLE 2
Analysis using different fluorescent dyes
Several fluorescent dyes for nucleic acid staining were compared
for applicability to homogeneous detection of PCR-SSP samples after
enzyme treatment; the results are shown in FIG. 3. Fluorescent dyes
ethidium bromide (A), ethidium homodimer (B), TO-PRO (C), TO-TO
(D), and YO-YO (E) were tested over a range of dye concentrations,
and each dye was tested using positive and negative PCR-SSP
samples. The positive samples contained DNA from a DR9 sample (cell
line DKB) amplified with DRBAMP-9 primers; the negative samples
contained the same DNA but were amplified with DRBAMP-1 primers
which were not complementary. For each dye concentration tested, 15
.mu.l of the amplified reactions were treated with 5 units of
Lambda exonuclease and 10 units of Exonuclease 1 . After enzyme
treatment, 1 .mu.l of dye was added to each well and the
fluorescence was measured. For EtBr and EthD, excitation was at 544
nm and emission was at 590 nm; for TO-PRO, TO-TO and YO-YO,
excitation was at 485 nm and emission was at 538 nm.
Although EtBr is the most common fluorescent dye used to visualize
DNA following gel electrophoresis, EtBr appeared to fluoresce even
in the absence of double stranded DNA, and the fluorescence ratios
between positive and negative samples were not easily
distinguished. EtHD had lower background fluorescence than EtBr,
but sensitivity for DNA detection was relatively poor.
Three relatively new highly fluorescent dyes, TO-PRO, TO-TO, and
YO-YO (23,24) were also compared. The best performance was seen
with YO-YO. Interestingly, this dye tended to lose fluorescence at
high dye/DNA ratios. At high concentrations, YO-YO was in a
suitable dye ratio for positive sample; for negative samples, the
fluorescence tended to decrease. This apparent quenching actually
contributed to an improvement in the signal difference between
positive and negative samples.
These results show that some of the recently developed dyes were
more sensitive and specific indicators for double stranded DNA than
the standard ethidium bromide dye when used in the instant method;
YO-YO had the best performance. This dye's fluorescence is low in
the absence of double stranded DNA and can increase about 3000-fold
when bound to double-stranded DNA (23,24). This ultra sensitive
nucleic acid stain, in combination with enzyme pretreatment,
improved the signal ratio between positive and negative samples and
allowed improved discrimination of HLA type.
EXAMPLE 3
Typing of clinical samples
The homogeneous detection method of the present invention was
applied to detect PCR-SSP amplification in seven different and
distinct clinical samples, and these samples were also typed by
both standard serological assays and by PCR methods using agarose
gel electrophoresis as the detection method. As shown in Table 3,
the homogeneous detection method could distinguish the correct
HLA-DR type for each specimen [positive fluorescence readings are
noted in bold type]. However, the results of the analysis according
to the present invention were obtained within about 30 minutes of
completion of the PCR amplification. For most samples, the
fluorescence was 5 to 20-fold greater in the positive PCR reaction
than in the negative reactions.
In a subset of the reactions, significant
TABLE 3
__________________________________________________________________________
HLA-DR typing using fluorescent homogeneous detection Sample Sample
Sample Sample Sample Sample Sample Primer pair 1 2 3 4 5 6 7
__________________________________________________________________________
DRBAMP-1 120 109 136 1524 432 109 114 DRBAMP-2 3121 132 150 163 331
1926 176 DRBAMP-15 2293 116 139 135 267 1943 114 DRBAMP-16 101 111
213 134 451 114 165 DRBAMP-3 2470 132 204 125 266 103 167 DRBAMP-17
2881 2828.sup.a 3903.sup.a 267 3002.sup.a 179 1662.sup.a DRBAMP-18
142 156 121 164 689 143 195 DRBAMP-4 705 4689 108 4608 1032 303 220
DRBAMP-11 153 149 119 215 3146 142 1255 DRBAMP-12 181 150 4003 219
817 567 173 DRBAMP-7 220 199 4482 238 4031 242 185 DRBAMP-8 236 165
248 426 823 150 151 DRBAMP-9 269 588 262 263 271 1530 143 DRBAMP-10
474 195 130 237 563 200 200 DRBAMP-13A 118 1994 150 132 400 109
1726 DRBAMP-13B 223 224 370 212 473 222 208 DRBAMP-14A 189 639 628
134 303 460 155 DRBAMP-14B 222 157 96 132 481 495 116 DRBAMP-52
4841 2955 4521 206 4713 228 5057 DRBAMP-53 171 4451 3795 4583 5153
1238 170 HLA-DR type DR15(2) DR4 DR7 DR1 DR7 DR9 DR11 DR17(3) DR13
DR12 DR4 DR11 DR15(2) DR13 DR52 DR52 DR52 DR52 DR52 DR53 DR53 DR53
DR53 DR53
__________________________________________________________________________
.sup.a Cross amplification: DRBAMP17 primer have cross
amplification with DR11, DR12 and DR13. (N) = The broad HLADR
specificity.
background fluorescence was detectable. This background did not
affect the accuracy of HLA typing in these specimens, and probably
was the result of sub-optimal primer design and resulting
primer-dimer formation and nonspecific amplification.
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__________________________________________________________________________
SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF
SEQUENCES: 40 (2) INFORMATION FOR SEQ ID NO:1: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:1: TTCTTGTGGCAGCTTAAGTTT21 (2)
INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:2: CCGCTGCACTGTGAAGCTCT20 (2) INFORMATION FOR SEQ ID
NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
CCTGTGGCAGCCTAAGAGG19 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORCANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:4: CCGCTGCACTGTGAAGCTCT20 (2)
INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORCANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:5: CCTGTGGCAGCCTAAGAGG19 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
TCCACCGCGGCCCGCGC17 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:7: CCTGTGGCAGCCTAAGAGG19 (2)
INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:8: ACCGCGGCGCGCCTGTCT18 (2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:10: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
GCAGTAGTTGTCCACCCGGC20 (2) INFORMATION FOR SEQ ID NO:11: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:12: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
AGCTCCGTCACCGCCCGGA19 (2) INFORMATION FOR SEQ ID NO:13: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:14: (i)
SEQUENCE CMMCTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic
acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE
TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:14: CTCCTGGTTATGGAAGTATCTC22 (2)
INFORMATION FOR SEQ ID NO:15: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:15: GTTTCTTGGAGCAGGTTAAA20 (2) INFORMATION FOR SEQ ID
NO:16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
CCGCTGCACTGTGAAGCTCT20 (2) INFORMATION FOR SEQ ID NO:17: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:18: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
CTGGCTGTTCCAGTACTCCT20 (2) INFORMATION FOR SEQ ID NO:19: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOLTRCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:
CACGTTTCTTGGAGTACTCTAC22
(2) INFORMATION FOR SEQ ID NO:20: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:20: GCTGTTCCAGGACTCGGCGA20 (2) INFORMATION FOR SEQ ID
NO:21: (i) SEQUENCE CMMCTERISTICS: (A) LENGTH: 20 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:
CCTGTGGCAGGGTAAGTATA20 (2) INFORMATION FOR SEQ ID NO:22: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HIA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
CCCGTAGTTGTGTCTGCACAC21 (2) INFORMATION FOR SEQ ID NO:23: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:
GTACTCTACGGGTGAGTGTT20 (2) INFORMATION FOR SEQ ID NO:24: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNFSS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:
CTGCAGTAGGTGTCCACCAG20 (2) INFORMATION FOR SEQ ID NO:25: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 17 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
CGGAGCGGGTGCGGTAT17 (2) INFORMATION FOR SEQ ID NO:26: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:26: CCCGTAGTTGTGTCTGCACAC21 (2)
INFORMATION FOR SEQ ID NO:27: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE, (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:27: CGGTTGCTGGAAAGACGCG19 (2) INFORMATION FOR SEQ ID
NO:28: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:
CCGCTGCACTGTGAAGCTCT20 (2) INFORMATION FOR SEQ ID NO:29: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:30: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
GTCCACCGCGGCCCGCTC18 (2) INFORMATION FOR SEQ ID NO:31: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:31: CACGTTTCTTGGAGTACTCTAC2 (2)
INFORMATION FOR SEQ ID NO:32: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORCANism: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:32: CTGTTCCAGTACTCGGCGCT20 (2) INFORMATION FOR SEQ ID
NO:33: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
CACGTTTCTTGGAGTACTCTAC22 (2) INFORMATION FOR SEQ ID NO:34: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
CCACCTCGGCCCGCCTCC18 (2) INFORMATION FOR SEQ ID NO:35: (i) SEQUENCE
CHARACTERISTICS: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:35: CACGTTTCTTGGAGTACTCTAC22 (2)
INFORMATION FOR SEQ ID NO:36: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOLTRCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:36: CACCGCGGCCCGCCTCTG18 (2) INFORMATION FOR SEQ ID
NO:37: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 22 base pairs (B)
TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear
(ii) MOLECULE TYPE-DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
CCCCAGCACGTTTCTTGGAGCT22 (2) INFORMATION FOR SEQ ID NO:38: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE:
nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii)
MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iv)
ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR
allele primers (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:
CCGCTGCACTGTGAAGCTCT20 (2) INFORMATION FOR SEQ ID NO:39: (i)
SEQUENCE CHARACTERISTICS: (A) LENGTH: 19 base pairs (B) TYPE:
nucleic acid
(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE:
DNA (genomic) (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi)
ORIGINAL SOURCE: (A) ORGANISM: HLA Class II DR allele primers (xi)
SEQUENCE DESCRIPTION: SEQ ID NO:39: AGCGAGTGTGGAACCTGAT19 (2)
INFORMATION FOR SEQ ID NO:40: (i) SEQUENCE CHARACTERISTICS: (A)
LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS:
single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii)
HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A)
ORGANISM: HLA Class II DR allele primers (xi) SEQUENCE DESCRIPTION:
SEQ ID NO:40: CTCCACAACCCCGTAGTTGTA21
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