Efficient linking of nucleic acid segments
AssigneeCity of Hope
InventorQiang Liu
Life of this patent
Estimating the term…
Abstract
Gene segment linking provides a simple, cost-effective way to produce an amplified DNA sequence containing linked segments of a single gene or multiple genes. The inventive method involves Multiplex PCR amplifications of gene sequences and linkage of the exons in a single reaction. Multiplex PCR of the gene segments is performed with primers having complementary tails which permit linkage of the gene segments and amplification of the linked gene in one polymerase chain reaction. The ability to construct an amplified DNA from genomic DNA containing several introns in two PCR steps allows the rapid production of DNA which heretofore required many time-consuming and expensive steps.
Claims54
A method of linking by PCR at least three DNA segments which occur in non-adjacent portions of target DNA wherein each DNA segment contains a sequence complementary to a sequence in the DNA segment or segments to which it is to be linked, comprising subjecting the at least three DNA segments to a linking PCR using
- a. a first primer which is complementary to the antisense strand of the first DNA segment to be linked and a second primer which is complementary to the sense strand of the last DNA segment to be linked;
- and
- b. at least one polymerase lacking 3′→5′ exonuclease activity and at least one polymerase containing 3′→5′ exonuclease activity.
A method of producing and amplifying DNA containing at least three linked DNA segments which occur in non-adjacent portions of target DNA, comprising
- a. providing a first primer and a second primer for each DNA segment to be amplified,
- i. the first primer (termed the D primer) having a 3′ portion which is complementary to the 3′ end of the antisense strand of the DNA segment and a 5′ tail which is complementary to the 5′ end of the second primer for the previous DNA segment;
- ii. the second primer (termed the U primer) having a 3′ portion which is complementary to the 3′ end of the sense strand of the DNA segment and a 5′ tail which is complementary to the 5′ end of the first primer for the subsequent segment;
- b. amplifying the at least three DNA segments by multiplex PCR using the pairs of first and second primers;
- and
- c. subjecting the at least three amplified DNA segments to a linking PCR using a first primer which is complementary to the antisense strand of the first DNA segment to be linked, a second primer which is complementary to the sense strand of the last DNA segment to be linked, at least one polymerase lacking 3′→5′ exonuclease activity and at least one polymerase containing 3′→5′ exonuclease activity.
A method of producing and amplifying DNA containing at least three linked DNA segments which occur in non-adjacent portions of target DNA, comprising
- a. providing a first primer and a second primer for each DNA segment to be amplified,
- i. the first primer (termed the D primer) having a 3′ portion which is complementary to the 3′ end of the antisense strand of the DNA segment and a 5′ tail which is complementary to a sequence internal to the previous DNA segment;
- ii. the second primer (termed the U primer) having a 3′ portion which is complementary to the 3′ end of the sense strand of the DNA segment and a 5′ tail which is complementary to a sequence internal to the subsequent segment;
- b. amplifying the at least three DNA segments by multiplex PCR using the pairs of first and second primers;
- and
- c. subjecting the at least three amplified DNA segments to a linking PCR using a first primer which is complementary to the antisense strand of the first DNA segment to be linked, a second primer which is complementary to the sense strand of the last DNA segment to be linked, at least one polymerase lacking 3′→5′ exonuclease activity and at least one polymerase containing 3′→5′ exonuclease activity.
Dependencies are inferred from the claim text.