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US5162209A
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Synthesis of full-length, double-stranded dna from a single-stranded linear dna template
Assignee: Beth Israel Deaconess Medical Center
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Filing: 1991-01-18
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Granted: 1992-11-10
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Expires: 2011-01-18
Abstract
Abstract A method for preparing a ds DNA from a ss DNA template, which method includes: (a) providing a first DNA strand; (b) adding a homopolymeric oligonucleotide tail to the 3' end of the first DNA strand, to yield a tailed first DNA strand; (c) providing a ss homopolymeric oligonucleotide primer complementary to a portion of the tail; (d) contacting the primer with the tailed first DNA strand; (e) synthesizing, in the presence of the primer and the tailed first DNA strand, a second DNA strand complementary to the first DNA strand; and (f) removing the tail and the primer from the first and second DNA strands, respectively; provided that one or both of the tail and the primer contain(s) RNA.
Inventors
- 👤 George Scheele
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-11-05 15:25
CPC Classifications
C; C12; C12N; C12N15/00; C12N15/09; C12N15/10; C12N15/1096; C12Q; C12Q1/00; C12Q1/68; C12Q1/6844; C12Q1/6853; C12Q1/6855
Claims Analysis
Total Claims: 9
Claim 1
Independent Claim
144 words
A method of making a ds DNA, said method comprising the following steps: (a) providing a first ss DNA strand; (b) adding to the 3' end of said first DNA strand a homopolmeric oligonucleotide tail comprising five deoxyribonucleotides, to yield a tailed first DNA strand; (c) providing a ss homopolymeric RNA primer complementary to a portion of said tail, said primer comprising five ribonucleotides; (d) contacting said primer with said tailed first DNA strand; (e) synthesizing, in the presence of said primer and said tailed first DNA strand, a second DNA strand complementary to said first DNA strand and having said primer at its 5' end; (f) after said synthesizing step, hydrolyzing said primer enzymatically or by the use of high pH, leaving said tail single-stranded; and (g) enzymatically removing said ss tail from said first DNA strand, to yield a full-length ds cDNA.
Claim 2
Independent Claim
144 words
A method of making a ds DNA, said method comprising the following steps: (a) providing a first ss DNA strand; (b) adding to the 3' end of said first DNA strand a homopolmeric oligonucleotide tail comprising five deoxyribonucleotides, to yield a tailed first DNA strand; (c) providing a ss homopolymeric RNA primer complementary to a portion of said tail, said primer comprising five ribonucleotides; (d) contacting said primer with said tailed first DNA strand; (e) synthesizing, in the presence of said primer and said tailed first DNA strand, a second DNA strand complementary to said first DNA strand and having said primer at its 5' end; (f) after said synthesizing step, hydrolyzing said primer enzymatically or by the use of high pH, leaving said tail single-stranded; and (g) enzymatically removing said ss tail from said first DNA strand, to yield a full-length ds cDNA.
Claim 3
Dependent Claim
34 words
A method of making a full-length, ds cDNA, said method comprising the method of claim 1; provided that said first DNA strand comprises a nucleotide sequence complementary to a strand of a naturally-occurring RNA.
Claim 4
Dependent Claim
38 words
A method of making a cDNA library, said method comprising the method of claim 2 and further comprising the step of inserting said full-length ds cDNA into a cloning vector, provided that said naturally-occurring RNA is an mRNA.
Claim 5
Dependent Claim
17 words
The method of claim 1, wherein (a) said tail comprises poly(dC), and (b) said primer comprises poly(rG).
Claim 6
Dependent Claim
17 words
The method of claim 1, wherein (a) said tail comprises poly(dG), and (b) said primer comprises poly(rC).
Claim 7
Dependent Claim
17 words
The method of claim 1, wherein (a) said tail comprises poly(dA), and (b) said primer comprises poly(rU).
Claim 8
Dependent Claim
17 words
The method of claim 1, wherein (a) said tail comprises poly(dT), and (b) said primer comprises poly(rA).
Claim 9
Dependent Claim
14 words
The method of claim 1, wherein said primer comprises between 5 and 30 nucleotides.
References Cited (Backward Citations)
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