Process for amplifying nucleic acid sequences
AssigneeCetus Corporation
InventorKary B. Mullis
Life of this patent
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Abstract
The present invention is directed to a process for amplifying any desired specific nucleic acid sequence contained in a nucleic acid or mixture thereof. The process comprises treating separate complementary strands of the nucleic acid with a molar excess of two oligonucleotide primers, and extending the primers to form complementary primer extension products which act as templates for synthesizing the desired nucleic acid sequence. The steps of the reaction may be carried out stepwise or simultaneously and can be repeated as often as desired.
Claims21
A process for amplifying at least one specific nucleic acid sequence contained in a nucleic acid or a mixture of nucleic acids wherein each nucleic acid consists of two separate complementary strands, of equal or unequal length, which process comprises:
- (a) treating the strands with two oligonucleotide primers, for each different specific sequence being amplified, under conditions such that for each different sequence being amplified an extension product of each primer is synthesized which is complementary to each nucleic acid strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
- (b) separating the primer extension products from the templates on which they were synthesized to produce single-stranded molecules;
- and
- (c) treating the single-stranded molecules generated from step (b) with the primers of step (a) under conditions that a primer extension product is synthesized using each of the single strands produced in step (b) as a template.
A process for amplifying a specific nucleic acid sequence contained in double-stranded DNA which process comprises:
- (a) separating the strands of the DNA by physical, chemical or enzymatic means;
- (b) treating the single strands with two oligodeoxyribonucleotide primers, in a molar excess of primer:
- its complementary strand, under conditions such that an extension product of each primer is synthesized, using E. coli DNA polymerase I or Klenow fragment thereof, which extension product is complementary to each DNA strand, wherein said primers are selected so as to be sufficiently complementary to different strands of each specific sequence to hybridize therewith such that the extension product synthesized from one primer, when it is separated from its complement, can serve as a template for synthesis of the extension product of the other primer;
- (c) separating the primer extension products from the templates on which they are synthesized to produce single-stranded molecules by physical, chemical or enzymatic means;
- and
- (d) treating the single-stranded molecules generated from step (c) with the two primers of step (b), in a molar excess of primer:
- its complementary molecule, under conditions such that a primer extension product is synthesized, using E. coli DNA polymerase I or Klenow fragment thereof, and using each of the single strands produced in step (c) as a template.
Dependencies are inferred from the claim text.