Variants of PCR Lecture 4 • Colony PCR • Multiplex PCR • Nested PCR • Reverse transcriptase PCR • Quantative PCR • Hot Start PCR • Touch down PCR Colony PCR • PCR screening of bacterial (E Coli) and yeast transformants containing cloned inserts • to determine insert size and/or orientation in the vector. • The presence of an insert and its size can be determined by growing each colony in liquid • Plasmid purified by a boiling or alkaline preparation protocol, • Digestion of the plasmid with restriction enzyme(s) that excises the insert • separation by agarose gel electrophoresis Multiplex PCR • several pairs of primers annealing to different target sequences. • This permits the simultaneous analysis of multiple targets in a single sample. • testing for genetic mutations, six or more amplifications might be combined • analysis of microsatellites and SNPs. Nested PCR • Two pairs of primers instead of one pair are used to amplify a fragment. • In the first PCR, one pair of primers is used to generate DNA products, which may contain products amplified from non-target areas. • Template (product of first PCR) in a second PCR, using one ('hemi-nesting') or two different primers whose binding sites are located (nested) within the first set, thus increasing specificity. Reverse transcriptase • detect RNA expression levels. qualitative study of gene expression. • detect gene expression through creation of complementary DNA (cDNA) transcripts from RNA. • quantification of RNA, by incorporating qPCR into the technique • qRT-PCR, RT-qPCR • most powerful, sensitive, and quantitative assay for the detection of RNA levels as compared to northern blot. • expression analysis of single or multiple genes • expression patterns for identifying infections and diseases. • May be either one step or two step RTPCR Real time or Quantative PCR • amplify and quantify targeted DNA molecule. • amplified DNA is detected as the reaction progresses in real time. • 1) non-specific fluorescent dyes that interclate with any double-stranded DNA • 2) sequence-specific DNA probes oligonucleotides labeled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target Applications rapidly detect nucleic acids that are diagnostic of, infectious diseases,cancer and genetic abnormalities. detect newly emerging diseases, such as new strains of flu in the fields of food safety, food spoilage and fermentation and for the microbial risk assessment of water quality. It is also used for the determination of zygosity of transgenic animals used in research Hot start PCR • Hot Start PCR avoids non-specific amplification of DNA by inactivating the taq polymerase at lower temperature. • dsDNA is denatured by heating the sample at its denaturing temperature and then the temperature is suddenly reduced to 55 degree C at which primer and Taq-polymerase is added • specific antibodies are used to block this Taq-polymerase at annealing temperature. • When the temperature raises for amplification to 72 degrees, the specific antibody detaches from Taq-polymerase and the amplification with greater specificity starts. Touchdown PCR • Primers will avoid amplifying nonspecific sequences. • The annealing temperature during a polymerase chain reaction determines the specificity of primer annealing. • The melting point of the primer sets the upper limit on annealing temperature. At temperatures just below this point, only very specific base pairing between the primer and the template will occur. • Touchdown PCR uses a cycling program annealing temperature is gradually reduced (e.g. 1-2°C /every second cycle). • The initial annealing temperature should be several degrees above the estimated Tm of the primers. • The annealing temperature is then gradually decreased until it reaches the calculated annealing temperature of the primers or some degrees below. Amplification is then continued.