1. An introduction of TAIL PCR
Speakers: Li Wing Yen Francisca,
Hau Pui Lei Benni,
Wong Fuk Ling

2. 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

3. 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

4. Advantages Simplicity High specificity High efficiency Speed
Less risks in chimeric artifacts
Direct sequencing
High sensitivity
Liu & Whittier, 1995

5. 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

6. 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

7. 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

8. 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.

9. 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.

10. 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

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

12. 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

13. 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)

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

15. 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)

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

17. 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

18. Cycling Orders
Liu & Whittier, 1995

19. Application

20. 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

21. P1 clones
Liu & Whittier, 1995

22. YAC clones
Liu & Whittier, 1995

23. BAC clones
Liu & Huang, 1998

24. 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

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

26. Direct sequencing
Liu & Whittier, 1995

27. 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

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

29. 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

30. 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.

31. 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

32. 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.

33. 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.

34. 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

35. 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.

36. Thanks for your attention!
The END
Thanks for your attention!