Transcript Principle of TAIL-PCR

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
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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
1)
2)
3)
4)
5)
6)
7)
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
Important features of TAIL-PCR
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Primer design
Annealing temperature
Cycling orders
Primer Design
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Specific primer (SP)
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•
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
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High-stringency cycle (thermal asymmetric)
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Reduced-stringency cycle (thermal
symmetric)
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Annealing temperature = 63oC
Annealing temperature = 44oC
Low-stringency cycle
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Annealing temperature = 30oC
Protocol of TAIL-PCR
AD primer
SP1 SP2 SP3
vector
insert
nontarget sequence
(A) Primary PCR with SP1 and AD
5 high stringency cycles
1 low stringency cycle
Protocol of TAIL-PCR
10 reduced stringency cycles
1 reduced stringency cycle
(thermal symmetric)
Product
yield:
TAIL-cycling
(12 super cycles)
2 high stringency cycles
(thermal asymmetric)
Specific product
(type I)
Nonspecific product
(type II)
Nonspecific product
(type III)
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
Specific product
Product
yield:
High (detectable)
Nonspecific product (type III)
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
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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
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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
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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
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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
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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 timeconsuming
Rapid isolation of promoter sequences
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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
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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
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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
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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
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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.