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US 6,607,899 B2Granted
Amplification-based cloning method
OwnerClarissa J. Chui, J. Christopher Grimaldi +3inventors
InventorsClarissa J. Chui, J. Christopher Grimaldi, Sean Milton, Minhong Yan, Sothy Yi
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Abstract
The invention provides a method for isolating a gene of interest from a cDNA library, which involves the use of an amplification step to amplify the gene of interest and an enrichment step to remove the template plasmids.
Claims22
A method of amplifying and isolating a nucleic acid molecule of interest from a mixture of nucleic acid molecules, comprising:
- (i) providing a recombinant nucleic acid library with a heterogeneous population of methylated, circular nucleic acid molecules as template molecules;
- (ii) annealing a first and a second primer to complementary strands of the circular nucleic acid molecule, to produce an annealed mixture, wherein
- the two primers extend in opposite directions relative to each other during polymerase chain reaction;
- the 5′ ends of the two primers are adjacent to each other;
- and
- wherein each primer is identical in sequence to its corresponding sequence in the nucleic acid molecule of interest;
- (iii) subjecting the annealed mixture to polymerase chain reaction, thereby producing an amplified mixture containing linear amplicons;
- (iv) digesting the amplified mixture with an enzyme that selectively cleaves methylated nucleic acid, thereby eliminating the template molecules and enriching the nucleic acid molecule of interest;
- and
- (v) isolating the nucleic acid molecule of interest.
7 more elements
A method of amplifying and isolating a nucleic acid molecule of interest from a recombinant nucleic acid library, comprising:
- (i) providing a recombinant nucleic acid library with a heterogeneous population of methylated, circular nucleic acid molecules as template molecules;
- (ii) annealing a first and a second primer to complementary strands of the circular nucleic acid molecule, to produce an annealed mixture, wherein
- the two primers extend in opposite directions relative to each other during polymerase chain reaction;
- the 5′ ends of the two primers are adjacent to each other;
- and
- wherein each primer is identical in sequence to its corresponding sequence in the nucleic acid molecule of interest;
- (iii) subjecting the annealed mixture to polymerase chain reaction, thereby producing an amplified mixture containing amplicons;
- (iv) ligating the amplicons in a ligation mix to produce circular replicons;
- (v) digesting the ligation mix with an enzyme that selectively cleaves methylated nucleic acid, thereby eliminating the template molecules and enriching the nucleic acid molecule of interest;
- (vi) transforming the replicons into a suitable host cell to generate transformed clones;
- and
- (vii) isolating the nucleic acid molecule of interest.
9 more elements
Dependencies are inferred from the claim text.