Method for sequencing both strands of a double stranded DNA in a single sequencing reaction

• US 6,251,610 B1
• Filed: 09/18/2000
• Issued: 06/26/2001
• Est. Priority Date: 09/08/1997
• Status: Expired due to Fees

• Current Assignee: Myriad Genetics
• Original Assignee: Myriad Genetics
• Inventors: Oliphant, Arnold, Gupte, Jamila
• Primary Examiner(s): Jones, W. Gary
• Assistant Examiner(s): Tung, Joyce
• Application Number:
  US09/664,560
• Time in Patent Office: 281 Days
• Field of Search: 435/91.2, 435/91.1, 435/6, 935/77, 935/78
• US Class Current: 435/6.11
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2535/119 Double strand sequencing

Abstract

A method is presented which uses a unique opposite strand joining strategy during PCR of an original DNA to generate a product which, when sequenced with a single sequencing primer yields the sequence of both strands of the original DNA. The PCR primers include 1) a modified oligomer corresponding to the 5′ end of a first strand of the DNA to be amplified wherein said modified oligomer includes the reverse complementary sequence to a sequence within said first strand of DNA and a specific PCR priming sequence which will specifically hybridize to a portion of the DNA to be amplified and 2) a second oligomer corresponding to the 5′ end of the second strand of the DNA to be amplified and which contains the priming sequence for the second strand of the DNA and will specifically hybridize to a portion of the DNA to be amplified. During PCR an intermediate product is formed where one end of one strand loops around to hybridize to its complement on the same strand. This results in a hairpin structure which elongates using its own strand as a template to form a double sized product that contains the sequence of both original strands. Upon denaturation this yields single strands with the single strands having the sequence of both of the original strands included in tandem. Sequencing these single strands using a single primer, e.g., a primer complementary to the second oligomer, yields the sequences of both strands of the DNA of interest.

Claims

1. A method effectively to join both strands of a double stranded DNA into a single strand of DNA, wherein said double-stranded DNA consists of a first strand and a second strand wherein said single strand of DNA comprises at least a portion of said first strand and said second strand, wherein said method comprises:

a) amplifying said double-stranded DNA to form amplified DNA using a pair of primers wherein one of said primers comprises a reverse complementary sequence of nucleotides;

b) denaturing said amplified DNA to form single-stranded DNA;

c) allowing intrastrand hybridization of said single-stranded DNA to occur; and

d) elongating said intrastrand hybridized single-stranded DNA to yield panhandle DNA which upon denaturation yields a single-stranded DNA comprising sequence of said both strands of said double stranded DNA.