PCR (Polymerase Chain Reaction Test) (cont.)
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What is RT-PCR?
RT-PCR is a PCR test that is designed to detect and measure RNA. Although initial PCR tests amplified DNA, many viruses and other biological components (for example, mitochondria) utilize RNA as their genetic material. RT-PCR differs from conventional PCR by first taking RNA and converting the RNA strand into a DNA strand. This is done by essentially the same method for PCR described above with the exception of using an enzyme termed reverse transcriptase instead of the DNA polymerase. The reverse transcriptase allows a single strand of RNA to be translated into a complementary strand of DNA. Once that reaction occurs, the routine PCR method can then be used to amplify the DNA. RT-PCR has been used to detect and study many RNA viruses.
RT-PCR should not be confused with another variation of PCR, termed Real-Time PCR. Real-Time PCR is a variation of PCR that allows analysis of the amplified DNA during the usual 40 cycles of the procedure. Although the procedure is similar to conventional PCR with cycling, Real-Time PCR uses fluorescent dyes attached to some of the building blocks or small nucleotide strands. Depending on the method used, fluorescence occurs when the amplified DNA strands are formed. The amount of fluorescence can be measured throughout the 40 cycles, and allows the investigators to measure specific products and their amounts during the amplification cycles. This often allows investigators or lab technicians to skip the gel electrophoresis or other secondary procedures needed for analysis of the PCR products, thus producing more rapid results.
Real-Time PCR and RT-PCR are variations or modifications of the original PCR test. However, there are many more variations (at least 25) that exist and are used to solve specific problems. They all have different names such as Assembly PCR, Hot-start PCR, Multiplex PCR, Solid-phase PCR and many others.
PCR is likely to continue to be modified to help answer many other questions in medicine, biology. and other fields of study.
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