A simplified procedure for cDNA and genomic library construction using nonpalindromic oligonucleotide adaptors.
The properties and characteristics of oligonucleotide adaptors for use in a simplified procedure for the construction of cDNA and genomic DNA libraries are described. The adaptors are suitable for joining to blunt ended cDNA or sheared genomic DNA, and then to the cohesive ends of restriction sites in vectors. Each adaptor consists of two oligonucleotides with complementary but nonpalindromic sequences that include an internal restriction site, a 5' phosphorylated blunt end, and an overlapping or staggered 5' hydroxylated end corresponding to a restriction endonuclease site in a vector of choice. Ligation of the blunt end to high molecular weight target DNA proceeds efficiently and there is no tandem concatenation of the adaptor. Insertion into the appropriate vector only requires ligation of the cohesive ends. There is no requirement for methylation, restriction enzyme cleavage, G-C tailing, or denaturation after ligation of the adaptor to the target DNA, all characteristics of other procedures.
D'Souza
C R
CR
Department of Biochemistry, Queen's University, Kingston, Ont., Canada.
Deugau
K V
KV
Spencer
J H
JH
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004247:DNA / Q000235:genetics*; D011088:DNA Ligases / Q000235:genetics; D005822:Genetic Vectors; D006168:Guinea Pigs; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010957:Plasmids
D015345:Oligonucleotide Probes; D004247:DNA; D011088:DNA Ligases