US 5,470,722 AGranted

Method for the amplification of unknown flanking DNA sequence

AssigneeUniversity of Iowa Research Foundation

InventorDouglas H. Jones

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Abstract

A method that permits the rapid amplification of unknown DNA that flanks a known site, such that one can walk into an uncharacterized region of DNA. In this method, human genomic DNA is restriction enzyme digested and then ligated to a 5' phosphorylated-oligonucleotide so that the 5' end of each strand of genomic DNA is extended and phosphorylated. The phosphorylated-oligonucleotide is constructed to render 5' end extensions that are complementary to the known sequence. Following denaturation and re-annealing under dilute conditions that promote intrastrand annealing and under high stringency, only those DNA strands containing the known sequence will form a stem-loop structure with a recessed and phosphorylated 5' end, rendering a substrate for a subsequent heat-stable ligation reaction to another oligonucleotide. This second oligonucleotide is complementary to the sequence immediately adjacent to the phosphorylated-oligonucleotide high stringency annealing site. The heat-stable ligation reaction appends a known sequence to the DNA segments containing the two known contiguous DNA sequences used for oligonucleotide annealing. This heat-stable ligation of known sequence permits the subsequent highly specific amplification of the unknown flanking DNA.

Claims29

Dependent claims

A method for the amplification of an unknown DNA sequence that flanks a known DNA sequence, comprising the steps of:

(a) digesting a double-stranded DNA fragment with a restricuion enzyme to yield 5' phosphorylated nucleotide overhang sequences, wherein said DNA fragment comprises a region of known DNA sequence and a region of unknown flanking DNA sequence to be amplified;
(b) annealing a 5' phosphorylated single-stranded oligonucieotide to a bridging-oligonucleotide to yield a double-stranded oligonucleotide with a 5' phosphorylated end;
(c) ligating said 5' phosphorylated single-stranded oligonucleotide of said double-stranded oligonucleotide whose non-phosphorylated 5' end is complementary to the cohesive ends generated in step (a) of said double-stranded DNA fragment to yield 5' phosphorylated nucleotide sequences complementary to a sequence portion within said known sequence region of said DNA fragment, wherein said sequence portion is an annealing site for said 5' phosphorylated nucleotide sequences;
(d) denaturing said 5' end-modified DNA fragment to produce single-stranded fragments containing said 5' phosphorylated nucleotide sequences;
(e) intra-strand annealing of a 5' phosphorylated nucleotide sequence to said annealing site within said known sequence region of a single-stranded fragment, wherein said single-stranded fragment is a fragment containing said annealing site located downstream (3') to said unknown flanking sequence region, to form a single-stranded loop, or pan portion, with a double-stranded stem of an otherwise single-stranded handle, of a panhandle structure;
f) heat stable ligating an oligonucleotide primer 1, complementary to a known sequence region downstream (3') from said annealing cite for said 5' phosphorylated nucleotide sequences, to the recessed 5' phosphorylated end of said double-stranded portion of said handle to elongate said double-stranded portion of said otherwise single-stranded handle of said panhandle structure;
and
(g) performing a first stage polymerace amplification reaction using a first set of oligonucleotide primers including said primer 1 annealing to said known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide sequences and a primer 2 complementary to a known sequence region both downstream (3') from said unknown flanking sequence region and upstream (5') from said annealing site for said 5' phosphorylated nucleotide sequences wherein initial priming by primer 2 occurs following annealing to said pan portion of said panhandle structure and is not inhibited by snapback annealing of said double-stranded handle of said panhandle structure.

5 more elements

A method for the amplification of an unknown DNA sequence that flanks a known DNA sequence, comprising the steps of:

(a) digesting a double-stranded DNA fragment with a restriction enzyme to yield 3' nucleotide overhang sequences, wherein said DNA fragment comprises a region of known DNA sequence and a region of unknown flanking DNA sequence to be amplified;
(b) ligating a 5' phosphorylated single-stranded oligonucleotide whose 3' end is complementary to the cohesive ends generated in step (a) of said double-stranded DNA fragment to yield 5' phosphorylated nucleotide overhang sequences complementary to a sequence portion within said known sequence region of said DNA fragment, wherein said sequence portion is an annealing site for said 5' phosphorylated nucleotide overhang sequences;
(c) denaturing said 5' end-modified DNA fragment to produce single-stranded fragments containing said 5' phosphorylated nucleotide sequences;
(d) intra-strand annealing of a 5' phosphorylated nucleotide overhang sequence to said annealing site within said known sequence region of a single-stranded fragment, wherein said single-stranded fragment is a fragment containing said annealing site located downstream (3') to said unknown flanking sequence region, to form a single-stranded loop, or pan portion, with a double-stranded stem of an otherwise single-stranded handle, of a panhandle structure;
(e) heat stable ligating an oligonucleotide primer 1, complementary to a known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide overhang sequences, to the recessed 5' phosphorylated end of said double-stranded portion of said handle to elongate said double-stranded portion of said otherwise single-stranded handle of said panhandle structure;
(f) performing a first stage polymerase amplification reaction using a first set of oligonucleotide primers including said primer 1 annealing to said known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide overhang sequences and a primer 2 complementary to a known sequence region both downstream (3') from said unknown flanking sequence region and upstream (5') from said annealing site for said 5' phosphorylated nucleotide sequences wherein inital priming by primer 2 occurs following annealing to said pan portion of said panhandle structure and is not inhibited by snapback annealing of said double-stranded handle of said panhandle structure;
and
(g) further performing a second stage polymerase amplification reaction to produce a nested primer product using a second set of oligonucleotide primers including a primer 3 complementary to both a portion of said known sequence region complementary to primer 1 and a portion of said annealing site for said 5' phosphorylated nucleotide sequences and a primer 4 complementary to a known sequence region both downstream (3') from said unknown flanking sequence region and upstream (5') from said known sequence region complementary to primer 2.

5 more elements

A method for the amplification of an unknown DNA sequence that flanks a known DNA sequence, comprising the steps of:

(a) digesting a double-stranded DNA fragment with a restriction enzyme to yield 5' phosphorylated nucleotide overhang sequences, wherein said DNA fragment comprises a region of known DNA sequence and a region of unknown flanking DNA sequence to be amplified;
(b) annealing a 5' phosphorylated single-stranded oligonucleotide to a bridging-oligonucleotide to yield a double-stranded oligonucleotide with a phosphorylated 5' end;
(c) ligating said 5' phosphorylated single-stranded oligonucleotide of said double-stranded oligonucleotide whose non-phosphorylated 5' end is complementary to the cohesive ends generated in step (a) of said double-stranded DNA fragment to yield 5' phosphorylated nucleotide sequences complementary to a sequence portion within said known sequence region of said DNA fragment, wherein said sequence portion is an annealing site for said 5' phosphorylated nucleotide sequences;
(d) denaturing said 5' end-modified DNA fragment to produce single-stranded fragments containing said 5' phosphorylated nucleotide sequences;
(e) intra-strand annealing of a 5' phosphorylated nucleotide sequence to said annealing site within said known sequence region of a single-stranded fragment, wherein said single-stranded fragment is a fragment containing said annealing site located downstream (3') to said unknown flanking sequence region, to form a single-stranded loop, or pan portion, with a double-stranded stem of an otherwise single-stranded handle, of a panhandle structure;
(f) heat stable ligating an oligonucleotide primer 1, complementary to a known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide sequences, to the recessed 5' phosphorylated end of said double-stranded portion of said handle to elongate said double-stranded portion of said otherwise single-stranded handle of said panhandle structure;
(g) performing a first stage polymerase amplification reaction using said oligonucleotide primer 1 annealing to said known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide sequences;
(h) further performing a second stage polymerase chain reaction using an oligonucleotide primer 3 complementary to both a portion of said known sequence region complementary to primer 1 and a portion of said annealing site for said 5' phosphorylated nucleotide sequences;
and
(i) further performing a third stage polymerase chain reaction using an oligonucleotide primer complementary to said annealing site for said 5' phosphorylated nucleotide sequences.

7 more elements

A method for the amplification of an unknown DNA sequence that flanks a known DNA sequence, comprising the steps of:

(a) digesting a double-stranded DNA fragment with a restriction enzyme to yield 5' phosphorylated nucleotide overhang sequences, wherein said DNA fragment comprises a region of known DNA sequence and a region of unknown flanking DNA sequence to be amplified;
(b) annealing a 5' phosphorylated single-stranded oligonucleotide to a bridging-oligonucleotide to yield a double-stranded oligonucleotide with a phosphorylated 5' end;
(c) ligating said 5' phosphorylated single-stranded oligonucleotide of said double-stranded oligonucleotide whose non-phosphorylated 5' end is complementary to the cohesive ends generated in step (a) of said double-stranded DNA fragment to yield 5' phosphorylated nucleotide sequences complementary to a sequence portion within said known sequence region of said DNA fragment, wherein said sequence portion is an annealing site for said 5' phosphorylated nucleotide sequences;
(d) denaturing said 5' end-modified DNA fragment to produce single-stranded fragments containing said 5' phosphorylated nucleotide sequences;
(e) intra-strand annealing of a 5' phosphorylated nucleotide sequence to said annealing site within said known sequence region of a single-stranded fragment, wherein said single-stranded fragment is a fragment containing said annealing site located downstream (3') to said unknown flanking sequence region, to form a single-stranded loop, or pan portion, with a double-stranded stem of an otherwise single-stranded handle, of a panhandle structure;
(f) heat stable ligating an oligonucleotide primer 1, complementary to a known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide sequences, to the recessed 5' phosphorylated end of said double-stranded portion of said handle to elongate said double-stranded portion of said otherwise single-stranded handle of said panhandle structure;
(g) performing a first stage polymerase amplification reaction using said oligonucleotide primer 1 annealing to said known sequence region downstream (3') from said annealing site for said 5' phosphorylated nucleotide sequences;
and
(h) further performing a second stage polymerase chain reaction using an oligonucleotide primer complementary to said annealing site for said 5' phosphorylated nucleotide sequences.

6 more elements

Dependencies are inferred from the claim text.