the principle of TAIL-PCR.pptVIP

An introduction of TAIL PCR Speakers: Li Wing Yen Francisca, Hau Pui Lei Benni, Wong Fuk Ling Contents of presentation Introduction What is TAIL PCR Advantages Principle of the TAIL PCR Details of TAIL PCR Application How to apply TAIL PCR in genome-relate research Thermal Asymmetric Interlaced (TAIL) PCR A simple and powerful tool for the recovery of DNA fragments adjacent to known sequences Was developed by Liu and Whittier in 1995 Utilizes a set of nested sequence-specific primers together with a shorter arbitrary degenerate (AD) primer The relative amplification efficiencies of specific and nonspecific products can be thermally controlled Advantages Simplicity High specificity High efficiency Speed Less risks in chimeric artifacts Direct sequencing High sensitivity 1) Simplicity neither special DNA manipulations before PCR (restriction digestion, ligation, etc) nor laborious screening afterward (Southern hybridization, primer labelling and extension, gel excision, etc) simple agarose gel analysis can confirm product specificity the requirement for the template DNA quantity (~ng) and purity are extremely modest 2) High specificity the proportion of coamplified nonspecific products is very low 3) High efficiency 60-80% of reactions yielded specific products with any given AD primer 4) Speed The successive amplification reactions can all be completed in 1 day 5) Less risks in chimeric artifacts TAIL PCR doesnt involve ligation step 6) Direct sequencing The high specific reaction products can be added directly to the sequencing reaction , no gel excision and purification are required 7) High sensitivity Single-copy sequences in genome can be amplified Principle of TAIL-PCR Important features of TAIL-PCR Primer design Annealing temperature Cycling orders Primer Design Specific primer (SP) Nested sequence specific primer complementary to vector sequence High melting temperature, Tm=58-63oC Arbitrary degenerate (AD) primer Relatively shorter Lower melti