Cold sensitive mutant DNA polymerases

Details Cold sensitive mutant DNA polymerases Cold sensitive mutant DNA polymerases

2000-06-06

2001-04-10

Fredman, Jeffrey (Department: 1655)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

C435S194000, C435S199000, C435S320100, C435S252800, C530S350000, C536S023700

Reexamination Certificate

active

06214557

ABSTRACT:

FIELD OF THE INVENTION
The present invention is directed to thermostable DNA polymerases, and more particularly, to novel mutants of Thermus aquaticus polymerases (Taq DNA polymerase). Specifically, the invention is directed to novel cold-sensitive mutants of Taq DNA polymerases and other thermostable DNA polymerases capable of catalyzing the amplification of polynucleotides by PCR (the polymerase chain reaction) and exhibiting substantially reduced activity at temperatures in the range from room temperature (25° C.) to 42° C. when compared to the same polymerase without at least one of the mutations, while retaining near-normal enzyme activity and functionality at the normal optimum temperature for the enzyme, 65 to 72° C. The present invention is also directed to nucleic acid and amino acid sequences encoding such mutants of Taq DNA polymerases, and vector plasmids and host cells suitable for the expression of these DNA sequences. Also described herein is an improved method which provides for an automatic hot start for performing polymerase chain reaction (PCR) amplification and other genetic analyses and manipulations using the DNA polymerases of the invention.
BACKGROUND OF THE INVENTION
PCR is a rapid and simple method for specifically amplifying a target DNA sequence in an exponential manner. Saiki, et al.
Science
239:487-4391 (1988). Briefly, the method as now commonly practiced utilizes a pair of primers that have nucleotide sequences complementary to the DNA which flanks the target sequence. The primers are mixed with a solution containing the target DNA (the template), a DNA polymerase and dNTPS for all four deoxynucleotides (adenosine (A), tyrosine (T), cytosine (C) and guanine(G)). The mix is then heated to a temperature sufficient to separate the two complementary strands of DNA. The mix is next cooled to a temperature sufficient to allow the primers to specifically anneal to sequences flanking the gene or sequence of interest. The temperature of the reaction mixture is then set to the optimum for the thermophilic DNA polymerase to allow DNA synthesis (extension) to proceed. The temperature regimen is then repeated to constitute each amplification cycle. Thus, PCR consists of multiple cycles of DNA melting, annealing and extension. Twenty replication cycles can yield up to a million-fold amplification of the target DNA sequence. In some applications a single primer sequence functions to prime at both ends of the target, but this only works efficiently if the primer is not too long in length. In some applications several pairs of primers are employed in a process commonly known as multiplex PCR.
The ability to amplify a target DNA molecule by PCR has applications in various areas of technology e.g., environmental and food microbiology (Wernars et al.,
Appl. Env. Microbiol
., 57:1914-1919 (1991); Hill and Keasler,
Int. J. Food Microbiol
., 12:67-75 (1991)), clinical microbiology (Wages et al.
J. Med. Virol
., 33:58-63 (1991); Sacramento et al.,
Mol. Cell Probes
, 5:229-240 (1991)), oncology (Kumar and Barbacid, Oncogene, 3:647-651 (1988); McCormick,
Cancer Cells
, 1:56-61 (1989)), genetic disease prognosis (Handyside et al.,
Nature
, 344:768-770 (1990)), and blood banking and forensics (
Jackson, Transfusion
, 30:51-57 (1990)).
DNA polymerase obtained from the hot springs bacterium
Thermus aquaticus
(Taq DNA polymerase) has been instrumental in DNA amplification, DNA sequencing, and in related DNA primer extension techniques. The DNA and amino acid sequences described by Lawyer et al.,
J. Biol. Chem
., 264:6427 (1989), GenBank Accession No. J04639, define the gene encoding
Thermus aquaticus
DNA polymerase and the enzyme
Thermus aquaticus
DNA polymerase as those terms are used herein. The highly similar DNA polymerase (Tfl DNA polymerase) expressed by the closely related bacterium
Thermus flavus
is defined by the DNA and amino acid sequences described by Akhmetzjanov, A. A., and Vakhitov, V. A.,
Nucleic Acids Research
20: 5839 (1992), GenBank Accession No. X66105. These enzymes are representative of a family of DNA polymerases, also including
Thermus thermophilus
DNA polymerase, which are thermostable. These enzymes lack a 3′-exonuclease activity such as that which is effective for editing purposes in mesophilic DNA polymerases such as
E. coli
DNA polymerase I, and phages T7, T3, and T4 DNA polymerases. Thermostable DNA polymerases which exhibit editing function are generally found in thermophilic archaebacteria such as
Pyrococcus furiosus
. Related DNA polymerases of this class are commonly known as Pfu, Pwo, Pfx, Vent, or Deep Vent.
The availability of thermostable DNA polymerases such as Taq DNA polymerase has both simplified and improved PCR. Taq DNA polymerase is stable up to 95° C. and its use in PCR has eliminated the necessity of repetitive addition of temperature sensitive polymerases after each thermal cycle. Additionally, Taq DNA polymerase can extend DNA at higher temperatures which tends to prevent the non-specific annealing of primers and thus, has improved the specificity and sensitivity of PCR.
Although significant progress has been made in PCR technology, the amplification of non-target oligonucleotides due to side-reactions, such as mispriming on non-target background DNA, RNA, and/or the primers themselves, still presents a significant problem. This is especially true in diagnostic applications where PCR is carried out in a milieu containing complex background DNA while the target DNA may be present in a single copy (Chou et al.,
Nucleic Acid Res
., 20:1717-1723 (1992)).
The temperature at which Taq DNA polymerase exhibits highest activity is in the range 62-72° C.; however, significant activity is exhibited at room temperature, approximately 25° C. to 37° C. In a normal or “cold start,” the primers may prime DNA extension at non-specific sequences because the formation of only a few base pairs at the 3′-end of a primer can result in a stable priming complex. The result can be competitive or inhibitory products at the expense of the desired product. As an example of inhibitory product, structures consisting only of primer, sometimes called “primer dimers” are formed by the action of DNA polymerase on primers paired with each other, regardless of the true target template. The probability of undesirable primer-primer interactions increases with the number of primer pairs in the reaction, as with multiplex PCR. During PCR cycling, these non-specific extension products can compete with the desired target DNA.
Further, it has been determined that side reactions often occur when all reactants are mixed at ambient temperature before thermal cycling is initiated. One method for minimizing these side reactions is termed “hot start” PCR. Many PCR analyses, particularly the most demanding ones, benefit from a hot start. About 50% of all PCR reactions show improved yield and/or specificity if a hot start is employed, and in some cases a hot start is absolutely critical. These demanding PCR analyses include those which have very low copy numbers of target (such as 1 HIV genome per 10,000 cells), denatured DNA (many DNA extraction procedures include a boiling step, so that the template is single-stranded during reaction setup), or contaminated DNA e.g., DNA from soil or feces and/or DNA containing large amounts of RNA. However, current methods of achieving a hot start are tedious, expensive, and/or have other shortcomings.
Hot start PCR may be accomplished by various physical, chemical, or biochemical methods. In a physical hot start, the DNA polymerase or one or more reaction components that are essential for DNA polymerase activity is not allowed to contact the sample DNA until all the components required for the reaction are at a high temperature. The temperature must be high enough so that not even partial hybridization of the primers can occur at any locations other than the desired template location, in spite of the entire genome of the cell being available for non-specific partial hybridization of the primers. Thus, t

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