Method for ligating adaptors to nucleic acids which methods are

Details Method for ligating adaptors to nucleic acids which methods are Method for ligating adaptors to nucleic acids which methods are

1995-07-07

1998-08-04

Arthur, Lisa B.

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

435 9152, 935 77, 935 78, C12P 1934

Patent

active

057892067

ABSTRACT:
The present invention is directed to cloning the ends of genes. Traditionally it has been very difficult to recover the 5' end of a gene. The present invention greatly eases this problem. The invention is a variation on RACE and uses a combination of techniques. Specific genes are purified by using three enrichment steps--1) a polymerase chain reaction, 2) a hybrid capture step, and 3) a second polymerase chain reaction. The inclusion of the hybrid capture step results in a greater enrichment than occurs with RACE. The ends of the gene are retained by use of a novel technique of attaching adaptors at the ends of the nucleic. The 5' end of the gene is conserved by preparing a first strand of cDNA and ligating to this an adaptor which is partially double stranded wherein the overhang or single stranded region of the adaptor is degenerate which allows for a fraction of the adaptor population to hybridize with the first strand of cDNA at the 3' end of the cDNA. This hybridization holds the adaptor in conjunction with the cDNA during the ligation step thereby resulting in a highly efficient ligation. The unique portion of the adaptor is used to design an oligomer to prime the second strand synthesis. None of the 5' sequence is lost in this method thereby allowing for a greater possibility of recovering the extreme 5' end of the gene.

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