A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors Journal Article
| Authors: | Sartoris, S.; Cohen, E. B.; Lee, J. S. |
| Article Title: | A rapid and improved method for generating cDNA libraries in plasmid and phage lambda vectors |
| Abstract: | We have developed a fast and efficient procedure for generating cDNA libraries in plasmid or phage λ vectors. We used Mo-MuLV reverse transcriptase to synthesize the first strand and directly added Escherichia coli DNA polymerase I with RNase H to synthesize the second strand. A special advantage of our procedure is the use of oligodeoxynucleotide adapters to insert the cDNA into the vector, avoiding the use of methylating enzymes and subsequent digestion with massive amounts of restriction endonucleases. This also obviates the need to tail the cDNA molecules with homopolymers, simplifying subsequent procedures such as sequencing or transfer to other vectors. Finally, we have used a rapid screening procedure to isolate full-length clones with oligodeoxynucleotide probes recognizing conserved regions at the 5' termini of the mRNA. The system is ideal for cloning and analyzing polymorphic alleles of genes, such as those of the major histocompatibility complex. © 1987. |
| Keywords: | human cell; methodology; allele; heredity; genetic vectors; lymphatic system; cloning, molecular; dna; genetic engineering; rna, messenger; escherichia coli; plasmid; plasmids; radioisotope; hla-d antigens; oligodeoxynucleotide; complementary dna; genetic polymorphism; hla system; oligodeoxyribonucleotides; histocompatibility; transformation, genetic; dna library; bacteriophage lambda; allelic polymorphism; recombinant dna; adapters; human; support, u.s. gov't, p.h.s.; dna probe; rna-directed dna polymerase; blood and hemopoietic system; hla genes; oligodeoxynucleotide probes |
| Journal Title: | Gene |
| Volume: | 56 |
| Issue: | 2-3 |
| ISSN: | 0378-1119 |
| Publisher: | Elsevier Science, Inc. |
| Date Published: | 1987-01-01 |
| Start Page: | 301 |
| End Page: | 307 |
| Language: | English |
| DOI: | 10.1016/0378-1119(87)90148-x |
| PUBMED: | 2445632 |
| PROVIDER: | scopus |
| DOI/URL: | |
| Notes: | Article -- Export Date: 5 February 2021 -- Source: Scopus |
