PCR is a method of amplifying a target sequence of DNA. The development of PCR has revolutionized the ways in which both DNA and RNA can be studied. PCR provides a sensitive, selective, and extremely rapid means of amplifying any desired sequence of DNA. Specificity is based on the use of two oligonucleotide primers that hybridize to complementary sequences on opposite strands of DNA and flank the target sequence (Figure 1). The DNA sample is first heat denatured (>90°C) to separate the two strands of the template DNA containing the target sequence; the primers, added in excess, are allowed to anneal to the DNA (typically at 50-75°C) in order to generate the required template-primer complex. Subsequently, each strand is copied by a special DNA polymerase, starting at the primer sites in the presence of all four dXTPs. The two DNA strands each serve as a template for the synthesis of new DNA from the two primers. Repeated cycles of heat denaturation, annealing of the primers to their complementary sequences, and extension of the annealed primers with DNA polymerase result in the exponential amplification of DNA segments of defined length. Product DNA doubles with each PCR cycle. DNA synthesis is catalyzed by a heat-stable DNA polymerase purified from one of a number of different thermophilic bacteria, organisms that grow at 70° to 80°C. Thermostable DNA polymerases can withstand multiple, short incubations at more than 90°, temperatures required to completely denature DNA. These thermostable DNA polymerases have made automation of PCR possible.

Fig1. The polymerase chain reaction technique is used to amplify specific gene sequences. Double-stranded DNA is heated to separate it into individual strands. These bind two distinct primers that are directed at specific sequences on opposite strands and that define the segment to be amplified. DNA polymerase extends the primers in each direction and synthesizes two strands complementary to the original two. This cycle is repeated 30 or more times, giving an amplified product of defined length and sequence. Note that the four dXTPs and the two primers are present in excess to minimize the possibility that these components are limiting for polymerization/amplification. It is important to note though that as cycle number increases incorporation rates can drop, and mutation/ error rates can increase.

DNA sequences as short as 50 to 100 bp and as long as 10 kb can readily be amplified by PCR. Twenty cycles provide an amplification of 10⁶ (ie, 2²⁰) and 30 cycles, 10⁹ (2³⁰). Each cycle takes less than or equal to 5 to 10 minutes so that even large DNA molecules can be amplified fairly rapidly. Because of subtle differences in DNA sequence with each new PCR target, the exact conditions for amplification must be empirically optimized. The workhorse PCR technique is central to many DNA/RNA sequencing technologies. The PCR method allows the DNA in a single cell, hair follicle, or spermatozoon to be amplified and analyzed. Thus, the applications of PCR to forensic medicine are obvious. PCR is also used to: (1) detect and quantify infectious agents, especially latent viruses; (2) make accurate genetic diagnoses by producing DNA fragments for sub sequent DNA sequence analyses to identify mutations; (3) detect allelic polymorphisms ranging from single base pair changes to large and small indels and gene amplification; (4) establish precise tissue types for transplants; and (5) study evolution, using DNA from archeological samples (6) for quantitative RNA analyses after RNA copying and mRNA quantitation by the so-called RT PCR method (cDNA copies of mRNA generated by a retroviral reverse transcriptase) or (7) to score in vivo protein-DNA occupancy using chromatin immunoprecipitation assays (see later). New uses of PCR are developed every year.

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مواضيع ذات صلة

Abnormalities of Chromosome Structure

Impact of Genomic Variation

Variation in individual Genomes

Variation in gene Expression and its relevance to medicine

Allelic Imbalance in gene expression

Gene expression as the integration of Genomic and Epigenomic signals

Epigenetic and Epigenomic aspects of Gene expression

DNA Microarray Technology

RNA Splicing

Initiation of Transcription

Fundamentals of Gene Expression

Cell cycle checkpoints