Chemistry: molecular biology and microbiology – Micro-organism – tissue cell culture or enzyme using process... – Preparing compound containing saccharide radical
Reexamination Certificate
2001-08-02
2002-06-11
Horlick, Kenneth R. (Department: 1656)
Chemistry: molecular biology and microbiology
Micro-organism, tissue cell culture or enzyme using process...
Preparing compound containing saccharide radical
C536S023100, C536S024330
Reexamination Certificate
active
06403341
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to a novel method of performing hot start PCR reactions. Furthermore, the present invention relates to achieving a greater specificity of amplification of a target nucleic acid. Also provided in the present invention are reagents and kits for performing magnesium precipitate hot start PCR reactions.
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 thermostable DNA polymerase and deoxynucleoside triphosphates (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 optionally reset to the optimum for the thermostable 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)).
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 and/or RNA while the target DNA may be present at a very low level down to 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 of 62° to 72° C., however, significant activity is also exhibited in the range of 20° to 37° C. As a result, during standard PCR preparation at ambient temperatures, 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. Other examples of inhibitory products are “wrong bands” of various length, caused by mispriming on the template DNA. During PCR cycling, these non-specific extension products can compete with the desired target DNA and/or lead to misinterpretation of the assay.
Since these side reactions often occur during standard PCR preparation at ambient temperature, one method for minimizing these side reactions involves “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, the temperature must be high enough so that base pairing of the primers cannot occur at template (or contaminating template) locations with less than perfect or near-perfect homology. This safe starting temperature is typically in the range of 50° to 75° C. and typically is about 10° C. hotter than the annealing temperature used in the PCR.
One physical way a hot start can be achieved is by using a wax barrier, such as the method disclosed in U.S. Pat. Nos. 5,599,660 and 5,411,876. See also Hebert et al.,
Mol. Cell Probes,
7:249-252 (1993); Horton et al.,
Biotechniques,
16:42-43 (1994). Using such methods, the PCR reaction is set up in two layers separated by a 1 mm thick layer of paraffin wax which melts at about 56° C. There are several methods which may be used to separate the reaction components into two solutions. For instance, all of the DNA is added, with 1×buffer but no dNTPs and no DNA polymerase enzyme, in a volume of 25 ml. One drop of melted wax is added and the tubes are all heated to 60° C. for one minute to allow the melted wax to form a sealing layer after which the tubes are cooled so the wax solidifies. Then a 25 ml mixture containing 1×buffer, all of the dNTPs, and the enzyme is added to each reaction. Finally, 1 drop of oil is added, to make 4 total layers. As the thermal cycler protocol heats the tubes to the first melting step (approximately 95° C.), the wax melts and floats to mix with the oil layer, and the two aqueous layers mix by convection as the temperature cycles.
One common variation involving the use of a wax barrier is that the reaction components are assembled with no magnesium ions so that the DNA polymerase enzyme is inactive. The magnesium ion encased in a wax bead is then (or initially) added. A further modification of the wax barrier used in PCR reactions is disclosed in the U.S. Pat. No. 5,599,660. Alternatively, at least one biological or chemical reagent needed for PCR is mixed with a wax carrier, resulting in a reagent that is solid at room temperature. Thus, the addition of other PCR reagents does not activate the DNA polymerase due to the fact that one or some of the reagents are sequestered in the wax. However, upon heating or the addition of a solvent, the sequestered reagent(s) is/are released from the carrier wax and allowed to react with other soluble reagents, leading to the initiation of the PCR reaction. After the amplific
Barnes Wayne M.
Rowlyk Katherine R.
Barnes Wayne M.
Horlick Kenneth R.
Senniger Powers Leavitt & Roedel
Spiegler Alexander H.
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