Sintesis Cdna Current Protocols

PREPARATION OF INSERT DNA FROM MESSENGER RNA
Construction of cDNA libraries from mRNA requires a series of steps similar to those outlined in Figure 5.5.1. Conversion of mRNA into double-stranded DNA suitable for insertion into a vector requires the action of at least six different enzymes. This conversion is described in two separate units: (1) conversion of mRNA into double-stranded cDNA (UNIT5.5) and (2) methylation and addition of linkers to double-stranded cDNA (UNIT 5.6). After completion of the protocols described in these units, the double-stranded DNA should be suitable for insertion into a vector. Although the procedure is divided into two parts, there are several convenient stopping places as noted in the Commentaries of the two units.

SECTION III

AAAAAAAA

oligo (dT)primed, reverse transcription
AAAAAAAA TTTTTTTT

second-strand synthesis intermediate
AAAAAAAA TTTTTTTT

second-strand synthesis with RNase H, DNA polymerase I, E. coli ligase
AAAAAAAA TTTTTT

complete second-strand synthesis and blunt-ended with T4 DNA polymerase, RNase H, RNase A, E. coli ligase
AAAAAA TTTTTT

EcoRI methylation and ligation of EcoRI linkers
CH3 GGAATTCC GGAATTCCCCTTAAGG CCTTAAGG CH3
AAAAAAGGAATTCC GGAATTCC TTTTTTCCTTAAGG CCTTAAGG

digestion of linkers and size fractionation
CH3 AATTCC GG CH3 AAAAAAGG TTTTTTCCTTAA

cDNA is ready to be cloned

Figure 5.5.1 Outline of cDNA synthesis and preparation for insertion into a vector. Contributed by Lloyd B. Klickstein, Rachael L. Neve, Erica A. Golemis, and Jeno Gyuris
Current Protocols in Molecular Biology(1995) 5.5.1-5.5.14 Copyright © 2000 by John Wiley & Sons, Inc.

Construction of Recombinant DNA Libraries

5.5.1
Supplement 29

UNIT 5.5

Conversion of mRNA into Double-Stranded cDNA
Enzymatic conversion of mRNA into double-stranded insert DNA can be accomplished by a number of different procedures. All of them involve the action of reverse transcriptase and oligonucleotide-primedsynthesis of cDNA. After that, the procedures in common use diverge considerably. There are a number of methods for synthesizing the second strand and several procedures for producing suitable ends for making clonable DNA. The major goals of these procedures are to construct insert DNA that is as long as possible, with a high yield of conversion of mRNA into DNA that can ligate to vector DNA. Thefollowing protocols require only commercially available reagents and are usually successful in producing good cDNA libraries. The basic protocol describes a method for making blunt-ended cDNA that can then be ligated to linkers (UNIT 5.6) for subsequent cloning into a unique restriction site such as EcoRI. The alternate protocol is a variation that requires fewer enzymatic manipulations and allowsconstruction of directional cDNA libraries, which are particularly desirable when the goal is to generate expression cDNA libraries. The alternate protocol takes advantage of a linker-primer consisting of (in order from 3′ to 5′) an oligo(dT) primer, a restriction site for the XhoI endonuclease, and a (GA)20 repeat to protect the restriction site during generation of the blunt-ended cDNA. Theinternal XhoI sites on the individual cDNA molecules are protected by incorporation of 5-methyl-dCTP in the first-strand nucleotide mix. The resulting cDNAs having unique ends can be cloned into EcoRI/XhoI–digested vectors after ligation of EcoRI adaptors to the 5′ end and digestion by XhoI to release the 3′ XhoI sites that were incorporated into the cDNA by the linker-primer. These changes result in aconsiderably streamlined procedure that is substantially faster and easier than the basic protocol. The quality and yield of insert DNA are in large part determined by the quality of the mRNA. An important feature of these procedures is that the yield at each step determines the yield at subsequent steps. Monitoring the efficiency of each reaction and determining the yield at each step is thus...