An introduction of TAIL PCR

An introduction of TAIL PCR

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

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, T m =58-63 o C
• Arbitrary degenerate (AD) primer
– Relatively shorter
– Lower melting temperature, T m =47-48 o C

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

Protocol of TAIL-PCR
SP1 SP2 SP3
AD primer
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)
TAIL-cycling
(12 super cycles)
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
Specific product
Nonspecific product (type III)
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

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 10 9 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 timeconsuming

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 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

Application of TAIL PCR
• 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.

Advantages
1) Simplicity
2) High specificity
3) High efficiency
4) Speed
5) Less risks in chimeric artifacts
6) Direct sequencing
7) 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