A strategy to study gene polymorphism by direct sequence analysis of cosmid clones and amplified genomic DNA.
A two-step strategy is described here to rapidly analyze gene-sequence variation or polymorphism. First, DNA sequences flanking the coding region of the gene to be analyzed are determined directly from a cosmid clone, including the gene, using the modified T7 DNA polymerase and sequencing primers based on the cDNA sequence of the gene. Second, the identified gene-flanking sequences are used to design amplification primers for the polymerase chain reaction (PCR) to permit amplification of DNA segments of up to 1 kilobase in genomic DNA from multiple individuals. These amplified DNA segments are directly sequenced using the thermostable Taq DNA polymerase.
Levedakou
E N
EN
Division of Biology, California Institute of Technology, Pasadena 91125.
Landegren
U
U
Hood
L E
LE
D001483:Base Sequence; D001709:Biotechnology; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase; D006801:Humans; D016133:Polymerase Chain Reaction; D011110:Polymorphism, Genetic; D011948:Receptors, Antigen, T-Cell / Q000235:genetics; D016693:Receptors, Antigen, T-Cell, alpha-beta; D019914:Taq Polymerase
D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D004247:DNA; D019914:Taq Polymerase; D004259:DNA-Directed DNA Polymerase