An efficient and reliable method for cloning PCR-amplification products: a survey of point mutations in integrin cDNA.
A highly efficient, non-labor-intensive method for cloning DNA fragments produced by PCR amplification was used to carry out a rapid survey of potential point mutations in integrin alpha 6 cDNA from 17 different cell-type sources. The method includes glass powder purification of the PCR reaction mixture, followed by simultaneous treatment with T4 polynucleotide kinase and DNA polymerase I, and another glass powder purification. Sequences from multiple subclones of each cell type were readily generated, aligned and checked for mismatches. Several commonly used alternative procedures were compared for cloning efficiency and size-fidelity of inserted DNA fragments.
Starr
L
L
Department of Cell Biology, Scripps Research Institute, La Jolla, CA 92037.
Quaranta
V
V
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000737:chemistry* / Q000302:isolation & purification; D004256:DNA Polymerase I; D006801:Humans; D016023:Integrins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D051381:Rats; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D016023:Integrins; D004247:DNA; D011116:Polynucleotide 5'-Hydroxyl-Kinase; D004256:DNA Polymerase I