Cloning and direct sequencing from lambda cDNA libraries using the polymerase chain reaction: suppressin and the vasopressin receptor as models.
A strategy using the polymerase chain reaction (PCR) to screen a lambda gt11 pituitary cDNA library for cDNAs encoding suppressin, a putative anti-proliferative protein, and a putative vasopressin receptor is described. The use of this technique will facilitate the demonstration of e.g. the presence of "neuropeptide receptors" on cells of the lymphoid system, confirming the concept of "shared ligands and receptors" by the neuroendocrine and the immune system. Neither of the genes encoding the proteins of the present study have previously been cloned. The PCR-screening procedure requires sequence information from the gene of interest which permits the generation of complementary primers. These primers are then used in combination with lambda phage primers complementary to regions flanking the cloning site in a PCR to amplify cDNAs derived from the gene of interest. This novel screening procedure yields cDNA related to the gene of interest, including the largest clone present in the library. To confirm the utility of this technique for cDNA libraries, the library was also screened using traditional cDNA hybridization techniques. The largest clone obtained by screening the cDNA library with PCR was the same as that obtained by the conventional technique. Thus, the results of these studies show that the PCR method can be used instead of more conventional means to screen cDNA libraries. Lastly, we describe a protocol for directly sequencing PCR-amplified DNA using the same primers that are used for amplification. The combined use of these two strategies permits cloning and sequencing of cDNAs from lambda cDNA libraries in a fraction of the time required using traditional screening techniques, but with identical results.
LeBoeuf
R D
RD
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294-0005.
Green
M M
MM
Berecek
K H
KH
Swords
B H
BH
Blalock
J E
JE
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004274:DNA, Recombinant; D015723:Gene Library; D007166:Immunosuppressive Agents; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D051381:Rats; D011956:Receptors, Cell Surface / Q000235:genetics*; D013951:Thymus Hormones / Q000235:genetics*; D014667:Vasopressins
D004274:DNA, Recombinant; D007166:Immunosuppressive Agents; D011956:Receptors, Cell Surface; D013951:Thymus Hormones; C053750:suppressin; D014667:Vasopressins; D004247:DNA