1. Field of the Invention
The present invention relates to a process for amplifying nucleic acid sequences. More specifically, it relates to an improved process for producing nucleic acid sequences from a DNA or RNA template which may be purified, or may exist in a mixture of nucleic acids. The resulting nucleic acid sequences may be exact copies of the template, or may be modified.
2. Description of Related Art
In the past, methods have been employed for amplifying nucleic acid sequences wherein both strands of the nucleic acid sequence to be amplified are synthesized by the same method. Such methods are prone to limitations due to the nature of the enzymes utilized in these processes.
In U.S. Pat. Nos. 4,683,195 and 4,683,202, DNA or RNA is amplified by the polymerase chain reaction (PCR). These patents are incorporated herein by reference in their entirety. This method involves the hybridization of an oligonucleotide primer to the 5' end of each complementary strand of the double-stranded target nucleic acid. The primers are extended from the 3' end in a 5'.fwdarw.3' direction by a DNA polymerase which incorporates free nucleotides into a nucleic acid sequence complementary to each strand of the target nucleic acid. After dissociation of the extension products from the target nucleic acid strands, the extension products become target sequences for the next cycle. In order to obtain satisfactory amounts of the amplified DNA, repeated cycles must be carried out, between which cycles, the complementary DNA strands must be denatured under elevated temperatures.
Traditional polymerases used in this process, such as E. coli DNA polymerase I have the limitation of being inactivated at temperatures necessary for the denaturation of the complementary strands. Thus, between each cycle of synthesis by such polymerases and after the heat denaturation step, a fresh aliquot of enzyme must be added to the reaction mixture so that extension of the primer and synthesis of the complementary strand may occur in the following cycle. This additional step increases the time required for amplification and decreases the ease of amplification which requires multiple steps.
In recent years, thermostable DNA polymerases have been discovered and isolated from thermophilic organisms such as Thermus aquaticus. Such thermostable polymerases make it possible to add enzyme at the beginning of a series of synthesis and denaturation steps, without the need to add a new aliquot of enzyme after each denaturation step.
A potential problem associated with PCR is the hybridization of a primer sequence to regions of the DNA molecule not intended to be amplified. Generally these undesired hybridizations occur because the target sample contains, in addition to the target sequence itself, other sequences with some complementarity to the primer sequences. If the 3' terminal nucleotides of the primer molecule are successfully hybridized to a sequence other than the target sequence, it is possible that primer extension may be successfully initiated by the polymerase enzyme, leading to the generation of an extension product different from the desired target sequence. Under some circumstances, this extension product will undergo exponential amplification, and be erroneously thought to be the desired target sequence.
A method of detecting a specific nucleic acid sequence present in low copy in a mixture of nucleic acids, called ligase chain reaction (LCR), has also been described. European patent application 0 320 308 describes this method and is incorporated herein by reference in its entirety. Target nucleic acid in a sample is annealed to probes containing contiguous sequences. Upon hybridization, the probes are ligated to form detectable fused probes complementary to the original target nucleic acid. The fused probes are disassociated from the nucleic acid and serve as a template for further hybridizations and fusions of the probes, thus amplifying geometrically the nucleic acid to be detected. The method does not use DNA polymerase.
LCR has disadvantages due to the need for at least four separate oligonucleotide probes for amplification. It also requires that the entire sequence of the target nucleic acid be known. Further, background signal can be caused by target independent ligation of the probes. Since the third probe hybridizes to the first probe and the fourth probe hybridizes to the second probe, the probes, when added in excess, can easily form duplexes among themselves which can be ligated independently of the target nucleic acid.
European Application No. 0 439 182 which is incorporated herein in its entirety by reference discloses a method of improving LCR amplification by providing probes/primers which hybridize to the target nucleic acid wherein one end is modified such that ligation cannot occur until the modified end is corrected. One such modification is the placement of a small gap between the probes preventing ligation of the probes. The gap sequence of the target nucleic acid must be selected such that the DNA sequence is comprised of three or less different nucleotides from the four possible nucleotides. The fourth nucleotide must be the first base complementary to the 5' end of the adjacent probe. The gap is then filled using a DNA polymerase or reverse transcriptase to extend one or more of the probes in a 5' to 3' direction in a target dependent manner to render the probes ligatable. The reaction mixture omits the fourth deoxynucleoside triphosphate complementary to the base at the 5' end of the adjacent probe. Because this method requires that the gap chosen in the target nucleic acid only contains bases which are complementary to a maximum of three of the deoxynucleoside triphosphates, the method limits the location of the gap on the target nucleic acid and also limits the size of the gap. Further, the method requires four primers. The application also discloses a method of PCR amplification wherein one end of the primer is modified such that the primer is not extendable by a polymerase enzyme. When this modification is removed in a template dependent manner, the primer can be extended. However, this type of PCR requires an additional step of removal of the modification before extension can occur.
In view of the foregoing disadvantages attendant with prior art methods of amplifying nucleic acid sequences, it should be apparent that there exists a need in the art for a method in which the fidelity of amplified sequences can be increased, which allows for the detection of a particular nucleic acid strand, and which allows one to efficiently examine multiple alleles in a single series of amplification steps.