Dual RT procedure for cDNA synthesis

Details Dual RT procedure for cDNA synthesis Dual RT procedure for cDNA synthesis

1998-09-28

2002-06-18

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Micro-organism, tissue cell culture or enzyme using process...

Preparing compound containing saccharide radical

C435S006120, C435S091200, C435S069100, C435S320100, C435S242000, C435S440000

Reexamination Certificate

active

06406891

ABSTRACT:

BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the field of molecular biology. More particularly, the present invention provides an improved reverse transcription method that allows the synthesis of long cDNA species.
2. Description of Related Art
A number of methods have been employed over the years for the synthesis of complementary DNA (cDNA). All of these methods utilize a reverse transcriptase (RT) for first strand synthesis and either a DNA polymerase or reverse transcriptase for second strand synthesis.
One of the first methods for isolating high quality cDNA was described by Efstratiadis et al. (1976). These investigators took advantage of the fact that, for reasons not completely understood, a small percentage of single-stranded cDNA will form hairpin structures at their 3′ ends. The hairpin structure could be used for priming of second strand synthesis and the hairpin was subsequently digested by S1 nuclease prior to cloning into a vector. Insertion into the vector was accomplished by using terminal transferase to form complementary homopolymeric tails at the ends of the vector and the cDNA. Despite the usefulness of this approach there were several drawbacks. It was not clear that all cDNAs formed hairpin structures and thus these libraries may not have been completely representational. Also, some degradation of the cDNA may result from the S1 nuclease digestion.
A major advance in the preparation of cDNA was the replacement synthesis method for second strand synthesis first introduced by Okayama and Berg (1982) and later modified by Gubler and Hofiman (1983). In this method, the second strand is synthesized by a nick translation procedure in which the mRNA strand is nicked by RNase H producing primers that can be utilized by
E. coli
polymerase I. This method is very efficient and eliminates the need for a S1 nuclease reaction. It remains the method of choice for second strand cDNA synthesis. For first strand synthesis, the enzymes primarily used have been either from the Moloney Murine Leukemia Virus (MMLV) or the avian myeloblastosis virus (AMV). The AMV RT was somewhat preferred because its optimum temperature was 42° C. compared to 37° C. for the MMLV enzyme. However, recently, the MMLV gene has been mutated in order to eliminate the endogenous RNase H activity, and this modified enzyme referred to as Superscript RT (Gibco-BRL), is superior for the production of full-length cDNAs.
A major impediment to the production of full-length cDNAs by existing techniques has been the occurrence of secondary structure in the mRNA. These and perhaps other naturally occurring pause sites inhibit the progression of the reverse transcriptases, and thus prevent the synthesis of full-length first strand cDNA. A number of methods, including the use of methylmercury hydroxide to denature the mRNA, have been used to remove the secondary structure during first strand synthesis. However, these methods have not proven to be completely satisfactory. Methylmercury hydroxide, for example, in addition to being highly toxic, inhibits RTs to some extent.
Another method for eliminating secondary structure in mRNA is to perform first strand synthesis at higher temperatures. However, this method also is flawed because the half lives of the MMLV and AMV enzymes at high temperatures are significantly reduced. Recently, however, RTs that are active at extremely high temperatures have been isolated. Unfortunately, such enzymes are not highly processive and therefore are not sufficient for the synthesis of full-length first strand cDNA.
An expression cloning approach that utilized an Epstein-Barr virus-based cloning vector capable of replicating extrachromasomally in human cells has been attempted to produce long cDNAs. The pEBS7 vector could be used for the efficient transformation and expression of cDNAs in human cells (Peterson and Legerski, 1991). Using a library prepared from mRNA derived from HeLa cells, the inventor's group was able to initially clone the gene that complements the xeroderma pigmentosum group C (XPC) gene (approximately 4 kb) (Legerski and Peterson, 1992). In addition, the cloning of the Cockayne's syndrome group A (CSA) gene (Tebbs et al., 1995), and a gene, XRCC3, that complements a Chinese hamster ovary (CHO) DNA repair mutant (Henning et al., 1995) also was achieved. Furthermore, two additional genes, XRCC2 and XRCC9, that complement CHO DNA repair mutants, have been cloned using the pEBS7 libraries.
Despite these successes, it remains apparent that very long cDNAs, above five or six kb, still were not well represented in these libraries. All of the genes discussed above were four kb or less in length. Attempts to clone longer gene sequences by this method have been unsuccessful. This defines a deficiency in the art in the production of full length cDNAs that has yet to be addressed.
SUMMARY OF THE INVENTION
In a particular embodiment, the present invention provides a method for the synthesis of cDNA comprising the steps of (a) providing a reaction mixture comprising a poly (A)+RNA, an oligonucleotide primer, dNTPs, (b) incubating the reaction mixture of step (a) with a highly processive enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a normal temperature range to allow first strand synthesis; (c) incubating the reaction mixture of step (b) with a thermostable enzyme composition having reverse transcriptase activity and incubating the reaction mixture at a temperature that inhibits the presence of secondary mRNA structures to generate a first strand; (d) adding the first strand to a reaction mixture for the synthesis of a second strand complementary to the first strand wherein the second strand synthesis reaction mixture comprises dNTPs and a DNA polymerase to initiate synthesis of the second strand and incubating the reaction mixture under conditions to allow the formation of a double-stranded cDNA. In specific embodiments, steps b and c are repeated. Steps b and c may be repeated once, twice, three, four or more times. More particularly, steps b and c are repeated until the appropriate length of first strand of the cDNA is generated.
In specific embodiments, the reaction mixture of step (a) further may comprise an RNase inhibitor. In other embodiments, the second strand synthesis reaction mixture of step (d) further comprises DEPC-treated H20. In still further embodiments, the second strand synthesis reaction mixture of step (d) further comprises RNase H. Certain embodiments further comprise the step of amplifying the double-stranded cDNA molecule of step (d). More particularly, the step of amplifying comprises PCR.
In specific embodiments, the temperature of step (b) is between about 37° C. and about 43° C. In other embodiments, the temperature of step (c) about 56° C. and about 95° C. The temperature in step (b) will be the temperature range optimal for any processive RT enzyme. The temperature range in step (c) will be any temperature range optimal for a thermostable RT. In specific examples, the processive reverse transcriptase may be selected from the group consisting of Superscript™; AMV Reverse Transcriptase, M-MLV Reverse Transcriptase. In particular examples, the thermostable reverse transcriptase is selected from the group consisting of Retrotherm™; Thermoscript™ and Tth reverse transcriptase.
In other embodiments, it is envisioned that the DNA polymerase is thermostable or non-thermostable. The DNA polymerase may be selected from the group consisting of DNA Polymerase I, T4 DNA Polymerase, DNA Polymerase I Klenow fragment, PLATINUM taq™. More particularly, the thermostable DNA polymerase may be selected from the group consisting of Tfl DNA Polymerase, Taq DNA Polymerase, Tli DNA Polymerase, Tth DNA Polymerase, Vent™, Deepvent™ and pfu.
In particularly defined embodiments, the sample comprises between about 0.1 and picograms and 10 micrograms of polyA RNA. Of course this is an exemplary range and other ranges of polyA RNA also are contemplat

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