Types of PCR.pptx

Types of PCR Multi-plex PCR More than one primer pair can be added to a PCR so that multiple amplifications are primed simultaneously, resulting in the formation of multiple products. Multiplex PCR is especially useful in typing or identification analy ses.  Multiplex PCR...

Types of PCR Multi-plex PCR More than one primer pair can be added to a PCR so that multiple amplifications are primed simultaneously, resulting in the formation of multiple products. Multiplex PCR is especially useful in typing or identification analy ses.  Multiplex PCR reagents and conditions require more complex optimization. Often, target sequences will not amplify with the same efficiency, and primers may interfere with other primers for binding to the target sequences. The conditions for the PCR must be adjusted for the optimal amplification of all products in the reaction. Reverse Transcriptase PCR Gene expression can be measured using a reverse transcription polymerase chain reaction (RT-PCR). RT- PCR is comprised of these steps: Conversion of RNA into cDNA using reverse transcriptase Amplification of cDNA using PCR  mRNA is more fragile than DNA and cannot be amplified by PCR. For that reason, mRNA is converted into complementary DNA (cDNA). cDNA is DNA that is synthesized from messenger RNA molecules. cDNA synthesis is catalyzed by an enzyme called reverse transcriptase, which uses RNA as a template for DNA synthesis. Reverse transcriptase was initially discovered and isolated from a retrovirus. These viruses contain an RNA genome; therefore the viruses need to produce a cDNA copy of their genome to be compatible with the host cell's molecular machinery. The RT-PCR reaction comprises the following steps: An oligo(T) fragment is used as a primer in order to bind to the 3’ poly(A)tail of each mRNA. Reverse transcriptase uses RNA as a template to synthesize cDNA strands. The resulting RNA-cDNA hybrid is separated by increasing the temperature. A gene-specific primer anneals to its complementary sequence. DNA polymerase produces the complementary DNA strand, starting from the primer binding site. The strands are separated by increasing the temperature and the PCR cycle is repeated. At the end of the RT-PCR reaction, there will be billions of copies of the gene of interest if the gene was expressed. The amount of DNA copies can be visualized by gel electrophoresis. Nested PCR Increased sensitivity offered by the PCR is very useful in clinical applications as clinical specimens are often limited in quantity and quality. The low level of target and the presence of interfering sequences can prevent a regular PCR from working with the reliability required for clinical applications. Real-Time (Quantitative) PCR or qPCR Real-time PCR, as the name suggests, measures the amount of PCR product at the end of each amplification cycle (i.e., in real time). To do this, you label the PCR products during or after the primer extension step of each amplification cycle. Let’s see how this is done. Examples of fluorescent dyes: SYBR Green, EtBr, etc Real-time PCR detection methods One uses DNA intercalating or minor groove targeting dyes that show enhanced fluorescence upon binding. With each amplification cycle, as the amount of the template DNA increases, the number of fluorophores bound, and therefore, the intensity of the fluorescent signal, increase. The second method uses fluorophore-labeled sequence- specific DNA probes  The most popular type uses the 5’-3’ exonuclease activity of Taq polymerase to chew up an oligonucleotide, which has a fluorophore and a quencher at the 5’- and 3’- ends respectively. The exonuclease activity of the polymerase helps separate the fluorophore from the quencher, which results in increased fluorescence.  Threshold cycle, or CT. The PCR cycle at which sample fluorescence crosses the threshold A plot of the accumulation of PCR product over 50 cycles of PCR (A) is a sigmoid curve. The generation of fluorescence occurs earlier with more starting template (solid lines) than with less (dotted lines). The cycle number at which fluorescence increases over a set amount, or fluorescence