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 Liu & Whittier, 1995

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 doesn't 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 After listening to Ling’s introduction, you may have some idea on TAIL-PCR. But actually how can we isolate the unknown DNA fragment flanking a known sequence by TAIL-PCR technique? And why we can get the specific products and excluding the non-specific one? Let me tell you the secret behind TAIL-PCR.

Important features of TAIL-PCR Primer design Annealing temperature Cycling orders There are three major factors leading to the success of this strategy. They are primer design, annealing temperature and cycling orders.

Primer Design Specific primer (SP) Arbitrary degenerate (AD) primer Nested sequence specific primer complementary to vector sequence High melting temperature, Tm=58-63oC Arbitrary degenerate (AD) primer Relatively shorter Lower melting temperature, Tm =47-48oC

Annealing Temperature High-stringency cycle (thermal asymmetric) Annealing temperature = 63oC Reduced-stringency cycle (thermal symmetric) Annealing temperature = 44oC Low-stringency cycle Annealing temperature = 30oC

Protocol of TAIL-PCR Primary PCR with SP1 and AD AD primer SP1 SP2 SP3 vector insert nontarget sequence Primary PCR with SP1 and AD 5 high stringency cycles 1 low stringency cycle

Protocol of TAIL-PCR 10 reduced stringency cycles TAIL-cycling (12 super cycles) 1 reduced stringency cycle (thermal symmetric) 2 high stringency cycles (thermal asymmetric) Specific product (type I) Nonspecific product (type II) Nonspecific product (type III) Product yield: High or middle (detectable or undetectable) High (detectable) Low (undetectable)

PCR Product of Primary Reaction Type II Type I Type III Liu & Whittier, 1995

Protocol of TAIL-PCR (B) Secondary PCR with SP2 and AD (10 super cycles) 1000-fold dilution of primary PCR product Nonspecific product (type III) Specific product Product yield: High (detectable) Very low (undetectable)

PCR Product of Secondary Reaction Type I Type II Type III Liu & Whittier, 1995

Protocol of TAIL-PCR (C) Tertiary PCR with SP3 and AD (20 normal cycles) 1000-fold dilution of secondary PCR product Specific product Agarose gel analysis Direct sequencing

Cycling Orders Liu & Whittier, 1995

Application

High efficiency to amplify insert end segments from P1, BAC and YAC clones TAIL-PCR as a powerful tool for amplifying insert end segments from P1, BAC and YAC clones The amplified products were highly specific and suitable as probes for library screening and as templates for direct sequencing The recover insert ends can also be used for chromosome walking and mapping

P1 clones Liu & Whittier, 1995

YAC clones Liu & Whittier, 1995

BAC clones Liu & Huang, 1998

High efficiency to amplify insert end segments from P1, BAC and YAC clones Many product bands from the primary TAIL-PCR reaction disappeared after the secondary TAIL-PCR, indicating that these were non-specific type II products Specific products were not always seen in the primary reactions due to their low concentration. However, these specific products becomes visible after the subsequent secondary reaction

Direct Sequencing Because it’s high specificity, unpurified TAIL-PCR products can be directly sequenced. Unpurified products yielded clear sequencing profiles

Direct sequencing Liu & Whittier, 1995

Recovery single-copy sequences from highly complex genome Amplification of single copy sequences was found technically more difficult in organisms with large genome. e. g. Inverse PCR is difficult to apply to genomes containing over 109 bp However, TAIL-PCR is very sensitive and can be applied to highly complex genomes

Recovery single-copy sequences from highly complex genome Liu , et al, 1995

Rapid isolation of promoter sequences The isolation of promoter and enhancer sequences is a crucial step in the study of the regulation of gene expression Flanking regions of genes, containing these elements, were conventionally isolated by screening genomic libraries using cDNA as probes, which is very time-consuming

Rapid isolation of promoter sequences Therefore, simpler and more reliable, and preferably PCR-based methods for promoter isolation are urgently required. Unlike Inverse PCR and ligation-mediated PCR, TAIL-PCR is a simple and efficient technique for genomic walking which does not require any restriction or ligation steps.

Rapid isolation of promoter seq. of Pal genes from yams Aligned DNA sequences of three TAIL PCR products obtained from the 5’-flanking regions of Pal genes of yams Terauchi & Kahl, 2000

Rapid isolation of promoter seq. of Pal from yams DNA sequences of PCR products overlapped perfectly with the 5’-end sequence of the cDNA. In the region isolated, a putative TATA box and several MREs could be identified.

Rapid isolation of promoter seq. of Pal genes from yams Isolated 5’-flanking regions of Pal and Pgi genes were fused to the GUS gene, and their activity was tested by transient transformation after delivery into tobacco BY2 cells by particle bombardment. All the isolated 5’-flanking regions were shown to drive reporter gene expression.

Conclusion TAIL-PCR is highly specific and efficient for amplification of DNA segments adjacent to known sequences Upon different modification, this technique could be used to handle vary tasks: Amplification of Insert Ends fragments from P1, YAC and BAC clones for chromosome walking

Conclusion Isolation of 5’ flanking region of genes Isolation of promoter sequences Isolation of T-DNA insert junctions for genome physical mapping, development of sequence-tagged sites (STS), and analysis of genomic sequences flanking T-DNA, transposon or ritrovirus insertions.

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