Amplification of mixed sequence nucleic acid fragments

• US 6,114,149 A
• Filed: 08/02/1991
• Issued: 09/05/2000
• Est. Priority Date: 07/26/1988
• Status: Expired due to Fees

• Current Assignee: LadaTech LLC
• Original Assignee: Genelabs Technologies, Inc. (GlaxoSmithKline PLC)
• Inventors: Fry, Kirk, Larrick, James, Tam, Albert
• Primary Examiner(s): Marschel, Ardin H.
• Application Number:
  US07/742,088
• Time in Patent Office: 3,322 Days
• Field of Search: 435/6, 435/91, 435/91.1, 435/91.2, 435/91.5, 435/91.51, 436/501, 536/27, 536/23.1, 536/25.3, 935/7, 935/16, 935/17, 935/78
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/1096 cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
• CPC Class Codes: C12Q 1/6809 Methods for determination or identification of nucleic acids involving differential detection
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2525/131 incorporating a restriction site
• CPC Class Codes: C12Q 2525/173 incorporating a polynucleotide run, e.g. polyAs, polyTs

Abstract

A method of amplifying a mixture of different-sequence DNA fragments which may be formed from RNA transcription, or derived from genomic single- or double-stranded DNA fragments. The fragments are treated with terminal deoxynucleotide transferase and a selected deoxynucleotide, to form a homopolymer tail at the 3'"'"' end of the anti-sense strands, and the sense strands are provided with a common 3'"'"'-end sequence. The fragments are mixed with a homopolymer primer which is homologous to the homopolymer tail of the anti-sense strands, and a defined-sequence primer which is homologous to the sense-strand common 3'"'"'-end sequence, with repeated cycles of fragment denaturation, annealing, and polymerization, to amplify the fragments. In one embodiment, the defined-sequence and homopolymer primers are the same, i.e., only one primer is used. The primers may contain selected restriction-site sequences, to provide directional restriction sites at the ends of the amplified fragments.

Claims

1. A method of amplifying a mixture of different-sequence double-stranded DNA fragments, comprising

treating the DNA fragments with terminal deoxynucleotide transferase and a selected deoxynucleotide triphosphate, to add a homopolymeric sequence to the 3'"'"' ends of both DNA fragment strands,

mixing the DNA fragments containing the homopolymeric sequence with (i) a homopolymer primer that is complementary to said homopolymeric sequence, (ii) heat-stable DNA polymerase and (iii) all four deoxynucleotide triphosphates,

heat denaturing the DNA fragment strands,

annealing the mixture under conditions to form DNA fragment/primer duplexes,

reacting the mixture under conditions in which the DNA fragment/primer duplexes are converted to double-stranded DNA molecules,

repeating said denaturing, annealing, and reacting steps until a desired degree of fragment amplification has been achieved.

2. The method of claim 1, wherein said treating further includes ligating a complementary primer to the 5'"'"' ends of both DNA fragment strands forming 5'"'"' common sequence ends, where said complementary primer is complementary to the homopolymeric sequence and can serve both as the homopolymer primer and a 5'"'"'-end common sequence primer.

3. The method of claim 1, for use in amplifying double-stranded DNA fragments:

(i) wherein said treating further includes,

(a) denaturing the double-stranded DNA fragments to yield single-strands,

(b) adding random primers, where said random primers contain (i) at their 3'"'"' end, random sequences effective to bind to complementary sequences of the DNA single-strands, and (ii) at their 5'"'"' ends, a common sequence, and

(c) annealing the mixture under conditions to form DNA single-strand/random primer duplexes, reacting the mixture under conditions in which the DNA single-strand/random primer duplexes are converted to double-stranded DNA molecules; and

(ii) where said mixing further includes a common sequence primer, where the sequence of the common sequence primer is the same as said common sequence.

4. The method of claim 3, where said treating further includes, protecting the 3'"'"'-ends of the duplex DNA fragments prior to said denaturing, to prevent homopolymer tailing of the original duplex fragments.

5. The method of claim 3, wherein the 5'"'"' common sequence and the 3'"'"' homopolymer sequence are complementary and, in said mixing, the common sequence primer and the homopolymer primer have the same sequence.

6. A method of amplifying a mixture of different-sequence nucleic acid fragment strands, comprising

manipulating the nucleic acid fragment strands to form a mixture of different sequence DNA fragment strands, where each DNA fragment strand has a 3'"'"'-end and further contains a 5'"'"'-end common sequence,

treating the DNA fragment strands with terminal deoxynucleotide transferase and a selected triphosphate, to add a homopolymeric sequence to the 3'"'"' ends,

mixing the DNA fragment strands containing the homopolymeric sequence with (i) a homopolymer primer that is complementary to said homopolymeric sequence, (ii) a common-sequence primer that has the same sequence as said common sequence, (iii) heat-stable DNA polymerase and (iv) all four deoxynucleotide triphosphates,

heat denaturing the DNA fragment strands,

annealing the mixture under conditions to form DNA fragment strand/primer duplexes,

reacting the mixture under conditions in which the DNA fragment strand/primer duplexes are converted to double-stranded DNA molecules,

repeating said denaturing, annealing, and reacting, until a desired degree of fragment amplification has been achieved.

7. The method of claim 6, wherein said manipulating includes (a) adding an oligo dT primer to a mixture of poly A RNA species and (b) transcribing the RNA in the presence of the oligo dT primer, reverse transcriptase, and all four deoxynucleotide triphosphates, wherein said 5'"'"'-end common sequence is the poly dT sequence.

8. The method of claim 7, wherein the homopolymeric region of the DNA fragment strands is non-complementary to said 5'"'"' poly dT common sequence.

9. The method of claim 8, for use in constructing a library of amplified double-stranded DNA inserts in a cloning vector and where the library construction further includes inserting the double-stranded DNA fragments in the vector between the DNA-insert sites.

10. The method of claim 7, wherein said homopolymeric sequence is a poly A sequence and in said mixing the common sequence primer and the homopolymer primer both contain poly dT sequences.

11. The method of claim 6, wherein said manipulating further includes

(a) isolating a mixture of RNA species,

(b) adding random primers having (i) at their 3'"'"' ends, random sequences effective to bind to complementary RNA sequences in the RNA species mixture, and (ii) at their 5'"'"' ends, said 5'"'"'-end common sequence, and

(c) under priming conditions, reacting the RNA species and attached random primers with reverse transcriptase and all four deoxynucleotide triphosphates, under conditions which produce DNA fragment strand copies of the RNA.

12. The method of claim 11, wherein the mixture of RNA species comprises RNA molecules of greater than about 3-8 kilobases in size.

13. The method of claim 11, wherein the 5'"'"' common sequence and the 3'"'"' homopolymer sequence are complementary and, in said mixing, the common sequence primer and the homopolymer primer have the same sequence.

14. The method of claim 11, wherein the 5'"'"' common sequence and the 3'"'"' homopolymer sequence are non-complementary and said homopolymer primer and common-sequence primer each have different 5'"'"'-end sequences.

15. A method of preparing a mixture of single-stranded RNA fragments for cloning in a cloning vector, comprising

transcribing the RNA fragments to an RNA/DNA complex using an RNA-strand primer, where said RNA-strand primer (i) has a sequence complementary to a 3'"'"' sequence of the RNA fragments and (ii) is effective to produce in the resulting RNA/DNA duplex DNA fragment strands having a selected common 5'"'"'-end sequence,

treating the DNA fragment strands with terminal deoxynucleotide transferase in the presence of a selected deoxynucleotide, to form a homopolymeric sequence at the 3'"'"'-end of the DNA fragment strands,

mixing the DNA fragment strands containing the homopolymeric sequence with (i) a homopolymer primer having a binding sequence which is complementary to the 3'"'"'-end homopolymeric sequence of the DNA fragment strands, (ii) a common-sequence primer which has the same sequence as said selected common 5'"'"'-end sequence, (iii) a heat-stable DNA polymerase and (iv) all four deoxynucleotide triphosphates,

heat denaturing the DNA fragment strands,

annealing the mixture under conditions to form DNA fragment stand/primer duplexes,

reacting the mixture under conditions in which the DNA fragment strand/primer duplexes are converted to double-stranded DNA molecules,

repeating said denaturing, annealing, and reacting steps until a desired degree of fragment amplification has been achieved.

16. The method of claim 15, wherein said RNA fragments are poly A RNAs, and said common-sequence primer includes a poly dT sequence.

17. The method of claim 15, wherein said transcribing is carried out with random primers having (i) at their 3'"'"' ends, random sequences effective to bind to complementary sequences of RNA fragments, and (ii) at their 5'"'"' ends, said 5'"'"'-end common sequence.