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US 2003/0207282 A1Published application
Method to amplify variable sequences without imposing primer sequences
AssigneeLos Alamos National Laboratory
InventorsAndrew M. Bradbury, Ahmet Zeytun
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Abstract
The present invention provides methods of amplifying target sequences without including regions flanking the target sequence in the amplified product or imposing amplification primer sequences on the amplified product. Also provided are methods of preparing a library from such amplified target sequences.
Claims43
A method of preparing an amplified product that is free of amplification primer sequences, said method comprising:
- (a) amplifying the target nucleic acid sequence in an amplification reaction comprising:
- (i) a first primer that specifically anneals to a region upstream of a target polynucleotide sequence, wherein the first primer comprises a first restriction enzyme recognition site that is recognized by a restriction enzyme that cleaves at a distance from the recognition site;
- and
- (ii) a second primer that specifically anneals to a region downstream of the target polynucleotide sequence, wherein the second primer comprises a second restriction enzyme recognition site that is recognized by a restriction enzyme that cleaves at a distance from the recognition site;
- and
- (b) cleaving said amplified product with the first and second restriction enzymes, thereby obtaining the amplified product that is free of amplification primer sequences.
3 more elements
A method of preparing a library, said method comprising the steps of:
- (a) preparing an amplified product that is free of amplification primer sequences by amplifying the target nucleic acid sequence in an amplification reaction comprising:
- (i) a first primer that specifically anneals to a region upstream of a target polynucleotide sequence, wherein the first primer comprises a first restriction enzyme recognition site that is recognized by a restriction enzyme that cleaves at a distance from the recognition site;
- and
- (ii) a second primer that specifically anneals to a region downstream of the target polynucleotide sequence, wherein the second primer comprises a second restriction enzyme recognition site that is recognized by a restriction enzyme that cleaves at a distance from the recognition site;
- (b) cleaving said amplified product with the first and second restriction enzymes, thereby obtaining the amplified product that is free of amplification primer sequences;
- and
- (c) linking said amplified product to a library vehicle.
4 more elements
Dependencies are inferred from the claim text.