Selective amplification of target polynucleotide sequences
AssigneeThe Board of Trustees of the Leland Stanford Junior University
InventorsJames Lawrence Burg, Philippe Jacques Pouletty, John Charles Boothroyd
Life of this patent
Estimating the term…
Abstract
A method is provided for multiplying the number of copies of a target polynucleotide sequence comprising a series of primer hybridization, extending, and denaturing steps to provide an intermediate double-stranded DNA molecule containing a promoter sequence (through the use of a promoter-sequence-containing primer) incorporated upstream from the target sequence. The double-stranded DNA intermediate is then used to grow multiple RNA copies of the target sequence. The resulting RNA copies can be used as target sequences to produce further copies. Multiple cycles of this sort can thereby exponentially increase the number of target sequence copies.
Claims56
A method for increasing the number of copies of a target sequence, or the complement of said target sequence, in a polynucleotide sequence having a 3′ region and a 5′ region, and said target sequence therebetween, comprising the steps of:
- (1) preparing a first double-stranded DNA comprising a first promoter sequence upstream from said target sequence by:
- (a) contacting an RNA polynucleotide sequence comprising a target sequence with a first primer able to bind to a region upstream of said target sequence;
- (b) extending said first primer with a DNA polymerase to form a primer extension product of said first primer having a sequence complementary to said target sequence,
- (c) making said primer extension product of said first primer available for hybridization with a second primer,
- (d) hybridizing said primer extension product of said first primer with a second primer able to bind to said primer extension product of said first primer at a region upstream of the sequence complementary to said target sequence, wherein one of said first and second primers comprises a 5′ promoter sequence,
- (e) extending said second primer, and if necessary said primer extension product of said first primer, with a DNA polymerase to prepare said first double-stranded DNA comprising a first promoter, and
- (2) transcribing multiple RNA copies from said first double-stranded DNA using a DNA-dependent RNA polymerase able to bind said promoter to thereby increase the number of copies of said target sequence, or the complement of said target sequence.
A method for increasing the number of copies of a target polynucleotide sequence or the complement of said target polynucleotide sequence which comprises:
- (1) sequentially hybridizing a target single-stranded polynucleotide molecule with primer sequences followed by extending said primers and denaturing, wherein at least one of said primers contains a promoter sequence, to produce a double-stranded DNA intermediate having a promoter sequence upstream from a target sequence;
- (2) growing multiple RNA copies of said target sequence from said intermediate using an RNA polymerase capable of binding said promoter;
- (3) preparing a second collection of double-stranded DNA intermediates having a promoter sequence upstream from said target sequence from said RNA copies by hybridizing said RNA copies with primer sequences, extending, and denaturing;
- and (4) transcribing multiple RNA copies from said second collection of double-stranded DNA intermediates.
The method of claim 34 wherein a labeled ribonucleoside triphosphate is provided as a substrate for one enzyme in said method.
A method for increasing the number of copies of a target sequence, or the complement of said target sequence, in a polynucleotide sequence having a 3′ region and a 5′ region, and said target sequence therebetween, comprising the steps of:
- (1) preparing a first double-stranded DNA comprising a first promoter sequence upstream from said target sequence by:
- (a) contacting a single stranded DNA polynucleotide sequence comprising a target sequence with a first primer able to bind to a region upstream of said target sequence,
- (b) extending said first primer with a DNA polymerase to form a primer extension product of said first primer having a sequence complementary to said target sequence,
- (c) making said primer extension product of said first primer available for hybridization with a second primer,
- (d) hybridizing said primer extension product of said first primer with a second primer able to bind to said primer extension product of said first primer at a region upstream of the sequence complementary to said target sequence, wherein one of said first and second primers comprises a 5′ promoter sequence,
- (e) extending said second primer, and if necessary said primer extension product of said first primer, with a DNA polymerase to prepare said first double-stranded DNA comprising a first promoter,
- (2) transcribing multiple RNA copies from said first double-stranded DNA using a DNA-dependent RNA polymerase able to bind said promoter to thereby increase the number of copies of said target sequence, or the complement of said target sequence.
- (3) preparing a population of second double-stranded DNA sequences comprising a second promoter by:
- (a) contacting said multiple RNA copies transcribed from said first double-stranded DNA with a third primer comprising a sequence able to bind to a region upstream of said target sequence, or the complement of said target sequence,
- (b) extending said third primer with an RNA dependent DNA polymerase to form a complementary DNA sequence comprising a primer extension product of said third primer comprising said target sequence or the complement of said target sequence,
- (c) making the primer extension product of said third primer available for hybridization with a fourth primer,
- (d) hybridizing said primer extension product of said third primer with a fourth primer able to bind to said primer extension product of said third primer at a region upstream of said target sequence, or the complement of said target sequence, wherein at least one of said third and fourth primers comprises a 5′ promoter sequence, and wherein said third and fourth primers may be the same or different from said first and second primers,
- (e) extending said fourth primer, and if necessary said primer extension product of said third primer, with a DNA polymerase to prepare said population of second double-stranded DNA comprising a second promoter, and
- (4) transcribing multiple RNA copies of said second double-stranded DNA using a DNA-dependent RNA polymerase able to bind said second promoter.
A method of detecting a target polynucleotide sequence in a sample, which comprises:
- multiplying the number of copies of said target sequence according to claim 46;
- and
- detecting the presence of said multiplied copies.
Dependencies are inferred from the claim text.