An introduction of TAIL PCR

Thermal Asymmetric Interlaced (TAIL) PCR

A simple and powerful tool for the recovery of DNA fragments adjacent to known sequencesWas developed by Liu and Whittier in 1995Utilizes a set of nested sequence-specific primers together with a shorter arbitrary degenerate (AD) primerThe relative amplification efficiencies of specific and nonspecific products can be thermally controlled

Principle of TAIL-PCR

Important features of TAIL-PCR

Primer designAnnealing temperatureCycling orders

Primer Design

Specific primer (SP)Nested sequence specific primer complementary to vector sequenceHigh 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 = 44oCLow-stringency cycle

Annealing temperature = 30oC

Protocol of TAIL-PCR

SP1 SP2 SP3AD primer

vector insert nontarget sequence

(A)Primary PCR with SP1 and AD

5 high stringency cycles

1 low stringency cycle

Protocol of TAIL-PCR10 reduced stringency cycles

2 high stringency cycles(thermal asymmetric)

1 reduced stringency cycle(thermal symmetric)

Nonspecific product(type II)

Specific product(type I)

Nonspecific product(type III)

Product yield:

High or middle(detectable or undetectable)

High(detectable)

Low(undetectable)

TAIL-cycling(12 super cycles)

PCR Product of Primary Reaction

Liu & Whittier, 1995

Type II

Type I

Type III

Protocol of TAIL-PCR

(B) Secondary PCR with SP2 and AD (10 super cycles)•1000-fold dilution of primary PCR product

Specific product Nonspecific product (type III)

Product yield:

High (detectable) Very low (undetectable)

PCR Product of Secondary Reaction

Liu & Whittier, 1995

Type III

Type II

Type I

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 clonesThe 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 productsSpecific 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

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 bpHowever, 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 expressionFlanking 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.

Conclusion

TAIL-PCR is highly specific and efficient for amplification of DNA segments adjacent to known sequencesUpon 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

Application of TAIL PCR

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

Advantages

1) Simplicity2) High specificity3) High efficiency 4) Speed5) Less risks in chimeric artifacts6) Direct sequencing7) High sensitivity

Liu & Whittier, 1995

1) Simplicityneither 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 specificitythe proportion of coamplified nonspecific products is very low

3) High efficiency60-80% of reactions yielded specific products with any given AD primer

4) SpeedThe successive amplification reactions can all be completed in 1 day

5) Less risks in chimeric artifactsTAIL PCR doesn't involve ligation step

6) Direct sequencingThe 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