Methods for DNA amplificating and sequencing

Details Methods for DNA amplificating and sequencing Methods for DNA amplificating and sequencing

2000-08-10

2002-08-06

Riley, Jezia (Department: 1656)

Chemistry: molecular biology and microbiology

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

Preparing compound containing saccharide radical

C435S006120, C435S091200

Reexamination Certificate

active

06428986

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates to a method for performing cycled primer extension on a DNA template, and more particularly to methods including a primer extension step such as polymerase chain reaction (PCR) amplification and nucleotide sequencing comprising performing said PCR and sequencing reactions in the presence of an osmoprotectant selected from proline 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine (hereinafter “THP(B)”), 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine (hereinafter “THP(A)”) and mixtures thereof, to improve yield and specificity of said reactions. The invention further relates to kits comprising proline, THP(B), THP(A), or mixtures thereof for use in PCR amplification and in cycle nucleotide sequencing.
BACKGROUND OF THE INVENTION
Primer extension on a DNA template is a step common to some of the most useful and powerful techniques in molecular biology. Polymerase chain reaction (PCR), one of these techniques, is a rapid, inexpensive and simple means of producing microgram amounts of DNA from minute quantities of source materials. Many variations on the basic procedure have now been described and applied to a range of disciplines.
In medicine, PCR's major impact is on the diagnosis and screening of genetic diseases and cancer, the rapid detection of mycobacteria and HIV, the detection of minimal residual disease in leukemia, and HLA typing. The PCR technique is also useful in forensic pathology and evolutionary biology, plays a central role in the human genome project and is routinely used in molecular biology processes (McPherson et al., 1992).
However, the practical use of PCR technology frequently faces difficulties and limitations. The necessity to convert originally duplex source DNA and then double-stranded DNA products into single stranded templates in every cycle of amplification is normally accomplished by thermal denaturation of DNA at 93-95° C. The DNA denaturation greatly depends on its nucleic base composition. A high GC content renders DNA amplification and sequencing very difficult, due to increased melting temperature and the stable secondary structure of the expanded motif. A common result of amplifying a region containing a repeat motif with a high GC content is the presence of additional amplification products, which do not correspond to the desired product (Varadaraj and Skinner, 1994). In addition, incomplete denaturation allows DNA strands to “snap back”, leading to a decrease in product yield. Denaturation steps that are conducted for long periods of time and/or at a high temperature lead to unnecessary loss of enzyme activity and dNTP decomposition.
Taq DNA polymerase, ordinarily used in PCR protocols, can withstand repeated exposure to the high temperature (94-95° C.) required for typical DNA strand separation, and thus simplifies the PCR procedure by eliminating the need to add an enzyme in each cycle. However, Taq polymerase appears to extend a mismatched primer/template in comparison to other polymerases with proofreading exonuclease activities, e.g. Klenow and T7 DNA polymerases, which are non-thermostable.
Another very effective technique employing primer extension is the cycle sequencing technique used for determining the order of nucleic acids in a target nucleotide sequence. This procedure involves repeated cycles of primer extension while the target nucleotide sequence is sequenced.
Similar considerations, as mentioned above for the PCR method, apply for the cycle sequencing procedure. In sequencing reactions as well, the complete denaturation of the template DNA is of crucial importance for a successful reaction. Thus, regions of DNA with repeat motifs, high GC content and rigid secondary structures are difficult to sequence. In addition, sequencing of a very long stretch of nucleotides, or of a target nucleotide sequence present in a minute amount is problematic. The ability to accomplish a complete denaturation of double stranded DNA and to perform sequencing reactions at reduced temperatures, either with Taq polymerase or with non-thermostable polymerase, is advantageous in terms of both yield and accuracy.
In an attempt to improve the yield and specificity of PCR and sequencing reactions, a number of buffer additives were employed. It was shown that certain cosolvents, such as DMSO (Pomp and Medrano, 1991; Filichkin and Gelvin, 1992), glycerol (Cheng et al., 1994; U.S. Pat. Nos. 5,432,065 and 5,545,539), formamide (Comey et al., 1991) and betaine (German Patents DE 4411594 C1 and DE 4411588 C1; Mytelka et al., 1996), facilitate standard PCR and/or cycle sequencing. It has been suggested that DMSO may affect the melting temperatures (Tm) of the template DNA and of the oligonucleotide primers and/or the degree of product strand separation at a particular “denaturation” as well as improving the thermal activity of Taq DNA polymerase (Gelfand and White, 1989). Glycerol may influence long amplifications by (i) doubling the thermal stability of Taq polymerase at 95-97° C., and (ii) effectively lowering DNA melting temperatures (by 2.5-3° C. for each 10% increase in glycerol concentration) (Cheng et al., 1994). Yet, the use of these buffer additives is limited, e.g. solutions containing glycerol in effective concentrations of 20-40% are viscous and difficult to handle (U.S. Pat. No. 5,432,065), DMSO in 10% concentration inhibits Taq DNA polymerase activity by 53% and T7 DNA polymerase is completely inactive in 40% formamide.
The compounds 2-methyl-4-carboxy-3,4,5,6-tetrahydropyrimidine [THP(B)], also known as ectoine, and its hydroxy derivative, 2-methyl-4-carboxy-5-hydroxy-3,4,5,6-tetrahydropyrimidine [THP(A)] were previously identified in the laboratory of the inventors of the present invention as metabolites in several
Streptomyces
microorganisms (Inbar and Lapidot, 1988a; 1988b and 1991; Malin and Lapidot, 1996). Ectoine was also found in a variety of halophilic and halotolerant bacteria (Galinski et al., 1985). THP(B) and THP(A) are zwitterionic compounds (Inbar et al. 1993;
FIG. 1
) with many useful properties such as osmoprotection and thermoprotection of several organisms of the
Streptomyces
species and
E. coli
cells (Malin and Lapidot, 1996). THP(B) and THP(A) are not toxic neither to mammalian cells nor to animals (Lapidot et al., 1995). Israel patent No. 100810 and corresponding U.S. Pat. No. 5,789,414 and European Patent No. EP 0553884 of the present applicants disclose that THP(A) and THP(B) interact with and protect DNA in non-tumor tissues from damage by DNA-binding drugs and thus can be used for decreasing the toxic effects of DNA-binding drugs such as adriamycin and actinomycin D.
Proline is another osmoprotectant that accumulates in plants, bacteria, algae and marine invertebrates as a response to salinity stress. Proline was shown to destabilize DNA and to partially counteract the effect of sodium chloride and spermidine on the stability of the double helix, and to lower the melting temperature of DNA in a concentration-dependent manner (Rajendrakumar et al., 1997).
None of the above references describes or suggests the use of proline, THP(A) or THP(B) or mixtures thereof as additives to PCR reaction mixtures and in reactions for nucleotide sequencing.
SUMMARY OF THE INVENTION
It has now been found, according to the present invention, that THP(B) is effective in lowering the melting temperature of double-stranded DNA, and that proline, THP(B) and THP(A) are capable of increasing the thermal stability of DNA polymerases at elevated temperatures, indicating that they can be useful in procedures involving melting of double-stranded DNA and/or polymerase-mediated DNA synthesis, such as in primer extension, in PCR (polymerase chain reaction) amplification and in DNA sequencing.
Thus, in one aspect, the present invention provides a method for performing a cycled primer extension reaction comprising the steps of:
(i) contacting a template DNA comprising a target sequence of nucleotides with at least one primer oligonucleotide complementary to a

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