U.S. patent application number 10/630333 was filed with the patent office on 2004-05-13 for simplified use of 5' ends of rnas for cloning and cdna library construction.
Invention is credited to Eberwine, James H., Madison, Roger.
Application Number | 20040091917 10/630333 |
Document ID | / |
Family ID | 21778077 |
Filed Date | 2004-05-13 |
United States Patent
Application |
20040091917 |
Kind Code |
A1 |
Eberwine, James H. ; et
al. |
May 13, 2004 |
Simplified use of 5' ends of RNAs for cloning and cDNA library
construction
Abstract
A method of directional cloning using the 5' ends of RNAs for
use, for example, in cloning and cDNA library construction is
provided.
Inventors: |
Eberwine, James H.;
(Philadelphia, PA) ; Madison, Roger; (Chapel Hill,
NC) |
Correspondence
Address: |
Licata & Tyrrell P.C.
66 E. Main Street
Marlton
NJ
08053
US
|
Family ID: |
21778077 |
Appl. No.: |
10/630333 |
Filed: |
July 30, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10630333 |
Jul 30, 2003 |
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09194513 |
Feb 25, 1999 |
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6623965 |
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09194513 |
Feb 25, 1999 |
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PCT/US97/06957 |
Apr 25, 1997 |
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60016617 |
May 1, 1996 |
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Current U.S.
Class: |
435/6.16 ;
435/91.2 |
Current CPC
Class: |
C12N 15/1096
20130101 |
Class at
Publication: |
435/006 ;
435/091.2 |
International
Class: |
C12Q 001/68; C12P
019/34 |
Claims
What is claimed:
1. An improved method of directional cloning of DNA comprising
annealing a first oligonucleotide encoding a restriction site to
single-stranded (-) cDNAs to create double-stranded regions on the
single-stranded (-) cDNA so that regions to be replicated in a
second (+) strand synthesis are limited.
2. The method of claim 1 further comprising annealing a second
oligonucleotide encoding the restriction site and having a
homopolymeric tail complementary to a homopolymeric tail at the 3'
end of the single-stranded (-) cDNAs to the 3' end of the
single-stranded (-) cDNA strand to form a stable replication
competent gapped single-stranded circle by hybridization of the
restriction site of the second oligonucleotide to the restriction
site of the single-stranded (-) cDNAs.
Description
[0001] This application is a continuation of U.S. Ser. No.
09/194,513 filed Feb. 25, 1999, which is the U.S. National Phase of
PCT/US97/06957 filed Apr. 25, 1997, which claims the benefit of
U.S. Provisional Application No. 60/016,617 filed May 1, 1996, each
of which are herein incorporated by reference in their
entireties.
BACKGROUND OF THE INVENTION
[0002] Cloning of DNA sequences encoding expressed proteins and
construction of cDNA libraries from poly A+ mRNAs isolated form
cells and tissues is currently performed in accordance with
procedures outlined in FIGS. 1a and 1b. However, the overall
process is very laborious and has several technical limitations.
Decreasing activity of the reverse transcriptase enzyme during
first strand synthesis of the reverse complementary DNA (-) strand
from the mRNA can result in yield of a product that is not full
length. In addition, truncations can occur during the second round
of synthesis to regenerate the corresponding "sense" coding (+) DNA
sequences from the (-) DNA strand. For abundant mRNAs, a complete
full length second strand is not always required as there is a
greater likelihood for overlapping cDNAs that span a complete
coding region. However, for smaller quantities of mRNA a full
length strand may be only represented a few times.
[0003] As outlined in FIGS. 1a and 1b, the current procedure also
requires homopolymeric tailing of both the cDNA sequences and the
restriction digested cloning vectors, thus doubling the amount of
manipulation involved. In addition, homopolymeric tailing of the
vector results in loss of the original restriction site thereby
limiting the ease of subsequent excision of the cloned cDNA region
for the transfer to other expression or amplification vectors.
[0004] Accordingly, improvements to simplify cloning of mRNA
sequences for use in the cloning of cDNAs for expression of
proteins and in the construction of cDNA libraries are desired.
SUMMARY OF THE INVENTION
[0005] In the present invention a simplified method of directional
cloning is provided. This method can be used, for example, in the
cloning of the 5' ends of cDNAs. The present invention differs from
prior art cloning methods requiring homopolymeric tailing of both
cDNA sequences and restriction enzyme digested vectors along with
complete second strand synthesis before homopolymeric tailing. The
method of the present invention improves the efficiency of the
cloning of 5'-cDNA ends thereby increasing the likelihood of
constructing full-length cDNA libraries comprised of overlapping
cDNA subsequences.
[0006] The present invention uses oligonucleotides encoding
restriction sites to create local double-stranded regions upon the
first strand cDNA product of reverse transcriptase. The
double-stranded regions are cleaved by double-strand requiring
restriction endonucleases and serve to limit the regions to be
replicated in a second (+) strand synthesis. Use of these
oligonucleotides also increases the accuracy of replication of the
entire shorter (-) cDNA strand to yield more of the 5' (+) cDNA
sequences necessary for obtaining a full representation of the
entire mRNA coding sequence.
[0007] The method of the present invention also uses an
oligonucleotide primer containing the same restriction site that is
homopolymerically tailed to complement the homopolymerically tailed
3' end of the (-) cDNA strand. The 5' end of this primer hybridizes
to the palindromic complement 3' end of the restriction digested
(-) cDNA strand thereby forming a more stable and replication
competent gapped single-stranded circle. The resultant
double-stranded product contains a unique copy of the targeting
restriction site encoded by the priming oligonucleotide. Cleavage
at this site yields double-stranded cDNA containing pairs that can
be directly ligated into appropriate multiple cloning sites of
commercial cloning and expression vectors. Since the restriction
site is preserved and flanks the cDNA insert, the desired cDNA
sequences can be readily excised and transferred to other vectors
if necessary.
BRIEF DESCRIPTION OF THE FIGURES
[0008] FIGS. 1a and 1b are schematics detailing the steps required
by prior art procedures used to obtain cDNA clones from poly A+
mRNA. A complete set of clones containing different cDNAs
representing all possible coding sequences derived from isolated
mRNAs constitute a cDNA library. Regions of RNA, first strand (-)
DNA and second strand (+) DNA are indicated by different stippling
patterns in the bars. The nucleotide sequences of homopolymeric
tailings are indicated in bold type. Steps are numbered
sequentially as indicated. Those listed on the left are for
preparation of cDNA. Those listed on the right are for preparation
of the cloning vector.
[0009] FIGS. 2a, 2b, and 2c are schematics detailing the steps of
the method of the present invention when used for obtaining cDNA
clones. The left portion of the figures show the method for cDNAs
derived from oligo-dT priming. The right portion of the figure
shows the method for random priming of the poly A+ mRNA.
DETAILED DESCRIPTION OF THE INVENTION
[0010] The present invention provides a method of directional
cloning which uses the 5'-ends of RNAs, for example, to obtain cDNA
clones. A detailed schematic of the method of the present invention
being used to produce cDNA clones is provided in FIGS. 2a-c. In
this embodiment, oligo-dT or random priming of poly A+ mRNA is used
to generate (-) first strand cDNAs. These cDNAs are then
homopolymerically tailed with dG or dC using terminal
deoxynucleotidyl transferase. After tailing, the heteroduplex is
denatured by heat and the mRNA removed by alkaline hydrolysis or
RNAse digestion to yield single-stranded (-) cDNA. The
single-stranded (-) cDNA is the mixed with an first oligonucleotide
incorporating a palindromic restriction site in the middle which is
flanked on both the 5' and 3' sides with at least two completely
degenerate nucleotides. In a preferred embodiment the first
oligonucleotide consists of ten bases, including a 6 base
palindromic restriction site, such as that for EcoRI, flanked by
two degenerative nucleotides, as the ten base overall length allows
a high degree of specificity of targeting with reasonable annealing
temperature. However, this oligonucleotide can be longer to
incorporate other palindromic restriction endonuclease recognition
sequences. Examples of restriction endonuclease recognition
sequences which can be used include, but are not limited to, BglII,
ClaI, EcoRV, SacI, KpnI, SmaI, BamHI, XbaI, SalI, AccI, AvaI, PstI,
SphI, HindIII, HincII, NsiI, NotI, SfiI, ApaI, NcoI, StuI, NdeI,
PvuII, and XhoI. After mixing, the oligonucleotide-cDNA mixture is
slowly cooled from 50.degree. C. to 37.degree. C. and the cognate
restriction enzyme is added. The resulting annealed, short
double-stranded DNA segments correspond to the positions of these
restriction sites on the (-) cDNA. Cleavage by the cognate
restriction enzyme yields single-stranded cDNAs bound on their 5'
end by the "sticky end" left by the restriction enzyme used and on
their 3' end by a poly-dG or -dC tract. Thus, the method of the
present invention allows specific site-directed cleavage of the
single-stranded (-) cDNA thereby eliminating the need for second
strand synthesis of the entire (+) cDNA to provide the
double-stranded restriction site as in prior art methods.
Accordingly, the present invention is much simpler and requires
less time than the prior art methods. Further, considerably smaller
amounts of oligonucleotide triphosphate reagents are required.
[0011] A second oligonucleotide comprising nucleotides
complementary to the 3' end of the cDNAs and containing the same
restriction site as in the first oligonucleotide is then annealed
to the 3' poly-dG or -dC tailed single-stranded (-) cDNA by a
similar cycle of heating and slow cooling as described above. Since
this single-stranded (-) cDNA contains the cognate "sticky end" at
its 5' terminus, the 5' end can loop back and also anneal to the
second oligonucleotide at the restriction site. The resulting
primed and gapped single-stranded (-) cDNA is stabilized and
rendered replication-competent for second strand synthesis of (+)
cDNA by the addition of a DNA polymerase and DNA ligase. Since the
cDNA region to be replicated is shorter than the original
full-length sequence, the likelihood of it being completely and
accurately replicated is increases over standard methods requiring
the traverse of a longer region of (-) cDNA. The resulting
double-stranded closed-circular cDNA is readily separated from
linear single-stranded fragments and trinucleotides by spin column
chromatography or agarose gel electrophoresis.
[0012] In addition, the resultant double-stranded cDNAs are readily
linearized with the cognate restriction enzymes to regenerate
"sticky ends": compatible for direct ligation into vectors
similarly linearized. This eliminates the extra effort in
homopolymeric tailing of the vector prior to insertion of the cDNA
by prior art methods. Further, the method of present invention
preserves the cloning restriction site thus allowing ready excision
of the desired cDNA sequences by the same restriction enzyme.
[0013] As will be obvious to those of skill in the art upon reading
this disclosure, the directional cloning method of the present can
also be used in different embodiments. For example, by utilizing a
complete set of oligonucleotides containing the common restriction
sites utilized in the multiple cloning sites of plasmid or phage
vectors, the present method can be used to provide a complete set
of restriction site delimited cDNA sublibraries which would greatly
facilitate both cloning and sequence analysis of cDNAs. A mRNA can
be effectively scanned for all potential restriction sites thereby
ensuring that a cDNA sublibrary would encode the corresponding
desired 5' ends for almost all encoded mRNA.
Sequence CWU 1
1
4 1 10 DNA Artificial sequence Synthetic 1 nngaattcnn 10 2 10 DNA
Artificial sequence Synthetic 2 nncttaagnn 10 3 11 DNA Artificial
sequence Synthetic 3 gaattccccc c 11 4 10 DNA Artificial sequence
Synthetic 4 aattcccccc 10
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