molecular biology

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2. DIFFERENT PCR
TECHNIQUES AND
THEIR APPLICATIONS

3. What is PCR
• It is a molecular technology aim to
amplify a single or few copies of the
DNA to thousands or millions of
copies.
• Developed in 1983 by Kary Mullis, PCR
is now a common and often
indispensable technique used in
medical and biological research labs
for a variety of applications. These
include diagnosis of infectious
diseases, DNA sequencing and DNA-
based phylogeny.
• In 1993, Mullis was awarded the Nobel
prize in Chemistry along with Michael
Smith for his work on PCR.
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5. Applications of PCR
Molecular Identification Sequencing Genetic Engineering
• Molecular Archaeology
• Molecular Epidemiology
• Molecular Ecology
• DNA fingerprinting
• Classification of organisms
• Genotyping
• Pre-natal diagnosis
• Mutation screening
• Drug discovery
• Genetic matching
• Detection of pathogens
• Bioinformatics
• Genomic cloning
• Human Genome Project
• Site-directed mutagenesis
• Gene expression studies
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7. ALLELE-SPECIFIC PCR
• A diagnostic or cloning technique which is based on
single-nucleotide polymorphisms (SNPs) (single-base
differences in DNA).
• It requires prior knowledge of a DNA sequence, including
differences between alleles, and uses primers whose 3'
ends encompass the SNP.
• PCR amplification under stringent conditions is much
less efficient in the presence of a mismatch between
template and primer, so successful amplification with an
SNP-specific primer signals presence of the specific SNP
in a sequence.
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8. Allele-Specific PCR flow
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9. ASYMMETRIC PCR
• This reaction preferentially amplifies one DNA strand in a
double-stranded DNA template.
• It is used in sequencing and hybridization probing where
amplification of only one of the two complementary strands
is required.
• PCR is carried out as usual, but with a great excess of the
primer for the strand targeted for amplification.
• Because of the slow (arithmetic) amplification later in the
reaction after the limiting primer has been used up, extra
cycles of PCR are required.
• A recent modification on this process, known as Linear-
After-The-Exponential-PCR (LATE-PCR), uses a limiting
primer with a higher melting temperature (Tm) than the
excess primer to maintain reaction efficiency as the limiting
primer concentration decreases mid-reaction . 98/14/2014

10. ASSEMBLY PCR OR POLYMERASE
CYCLING ASSEMBLY (PCA)
• This entails the artificial
synthesis of long DNA
sequences by performing PCR
on a pool of long
oligonucleotides with short
overlapping segments.
• The oligonucleotides alternate
between sense and antisense
directions, and the overlapping
segments determine the order
of the PCR fragments, thereby
selectively producing the final
long DNA product.
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11. Assembly PCR:
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12. HOT-START PCR
• A technique that reduces non-specific amplification
during the initial set up stages of the PCR.
• It may be performed manually by heating the reaction
components to the melting temperature (e.g., 95°C)
before adding the polymerase.
• Specialized enzyme systems have been developed that
inhibit the polymerase's activity at ambient temperature,
either by the binding of an antibody or by the presence of
covalently bound inhibitors that only dissociate after a
high-temperature activation step.
• Hot-start/cold-finish PCR is achieved with new hybrid
polymerases that are inactive at ambient temperature and
are instantly activated at elongation temperature.
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13. • Mechanical hot start PCR: all components of PCR
are added to the PCR vial except for the DNA
polymerase enzyme which will be added just at the
first denaturation step.
• Non mechanical hot start PCR: The use of a form
of Taq DNA polymerase, for example, Amplitaq Gold
which is activated only if the reaction mixture is
heated at about 94°C (the first denaturation step).
Other method depends on covalent linking of the
polymerase enzyme to certain inhibitors. The
enzyme becomes dissociated from these inhibitors at
the first denaturation step.
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14. Hot-Start PCR flow
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15. HELICASE-DEPENDENT AMPLIFICATION
•This PCR is similar to traditional PCR, but uses a
constant temperature rather than cycling
through denaturation and annealing/extension
cycles.
• DNA helicase, an enzyme that unwinds DNA, is
used in place of thermal denaturation.
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16. IN SITU PCR (ISH)
A polymerase chain reaction that actually takes place inside the
cell on a slide. In situ PCR amplification can be performed on
fixed tissue or cells.
• Uses
Detection and diagnosis of viruses and other infectious agents in
specific cell types within tissues.
Detection and characterization of tumor cells within tissue.
Detection and diagnosis of genetic mutations in inherited
diseases.
Detection of gene and gene expression in a tissue.
Any assay in which identification of cells expressing a target
gene is required. Main advantages are low background, high
specificity, fast assay with shorter turn-around time and no need
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18. INVERSE PCR
• The inverse PCR method includes a series of digestions
and self-ligations with the DNA being cut by a restriction
endonuclease. This cut results in a known sequence at
either end of unknown sequences.
• Inverse PCR uses standard PCR (polymerase chain
reaction), however it has the primers oriented in the
reverse direction of the usual orientation. The template
for the reverse primers is a restriction fragment that has
been ligated upon itself to form a circle.
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19. Inverse PCR Steps
• Target DNA is lightly cut into smaller fragments of several
kilobases by restriction endonuclease digestion.
• Self-ligation is induced under low concentrations causing the
phosphate backbone to reform. This gives a circular DNA ligation
product.
• Target DNA is then restriction digested with a known
endonuclease. This generates a cut within the known internal
sequence generating a linear product with known terminal
sequences. This can now be used for PCR (polymerase chain
reaction).
• Standard PCR is conducted with primers complementary to the
now known internal sequences.
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20. Figure: Inverse PCR Protocol 208/14/2014

21. USES OF INVERSE PCR
• It is commonly used to identify the flanking sequences
around genomic inserts.
• Inverse PCR has numerous applications in molecular
biology including the amplification and identification of
sequences, flanking transposable elements, and the
identification of genomic inserts.
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22. LIGATION-MEDIATED PCR
• Ligation-mediated PCR uses small DNA oligonucleotide 'linkers'
(or adaptors) that are first ligated to fragments of the target
DNA. PCR primers that anneal to the linker sequences are then
used to amplify the target fragments. This method is deployed
for DNA sequencing, genome walking, and DNA footprinting.
• The principle of Ligation Mediated PCR (LM-PCR).
1-Ligation with excess of primers,
2-Polymerase chain reaction of individual fragments.
In LM-PCR, each fragment is amplified independently so that due
to intrinsic differences among individual fragments, some
fragments are amplified less efficiently than others. This results
in non-uniform representation of original genetic material in
the resultant amplicon, which consequently leads to loss of
genetic information and inaccurate results.
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23. • Primer-extension step (Step 3): a gene-specific primer
(Primer 1) was annealed at 48°C and the primer was
extended with Sequenase enzyme at 48°C.
• Ligation step (Step 4): all extended DNA fragments with
a blunt-end and 5'-phosphate group were ligated to an
unphosphorylated synthetic asymmetric double-strand
linker.
• Linear amplification step (Step 5): a second gene-specific
primer (Primer 2) was annealed to DNA fragments for a
one-cycle extension using Taq DNA polymerase.
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24. • Exponential amplification step (Step 6): the primer 2 and
the linker primer (the longest of the two oligonucleotides
of the linker) were used to exponentially and specifically
amplify DNA fragments.
• Sequencing gel electrophoresis and electroblotting (Step
7): amplified DNA fragments were size-separated on a
denaturing 8% polyacrylamide gel and transferred onto a
nylon membrane by electroblotting.
• Hybridization (Step 8) the nylon membrane was
hybridized overnight with a gene-specific probe.
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26. USES OF LM PCR
• Ligation-Mediated Polymerase Chain Reaction (LMPCR)
is the most sensitive sequencing technique available to
map single-stranded DNA breaks at the nucleotide level
of resolution using genomic DNA. LMPCR has been
adapted to map DNA damage and reveal DNA–protein
interactions inside living cells.
• However, the sequence context (GC content), the global
break frequency and the current combination of DNA
polymerases used in LMPCR affect the quality of the
results.
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27. MULTIPLEX LIGATION-DEPENDENT PROBE
AMPLIFICATION (MLPA)
• MLPA is used to establish the copy number of up to 45
nucleic acid sequences in one single multiplex reaction.
The method can be used for genomic DNA (including
both copy number detection and methylation
quantification) as well as for mRNA profiling, it permits
multiple targets to be amplified with only a single primer
pair, thus avoiding the resolution limitations of multiplex
PCR.
• The principle of MLPA is based on the identification of
target sequences by hybridization of pairs of MLPA
probes that bind to adjacent sequences and can then be
joined by a ligation reaction. In order to make one copy
of each target sequence, specific MLPA probes are added
to a nucleic acid sample for each of the sequences of
interest.
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28. • The sequences are then simultaneously amplified with the
use of only one primer pair, resulting in a mixture of
amplification products, in which each PCR product of each
MLPA probe has a unique length.
• One PCR primer is fluorescently or isotopically labelled so
that the MLPA reaction products can be visualized when
electrophoresed on a capillary sequencer or a gel. Resulting
chromatograms show size-separated fragments ranging
from 130 to 490 bp.
• The peak area or peak height of each amplification product
reflects the relative copy number of that target sequence.
• Comparison of the electrophoresis profile of the tested
sample to that obtained with a control sample enables the
detection of deletions or duplications of genomic regions of
interest
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31. MULTIPLEX PCR
• Multiplex PCR is a widespread molecular biology technique for
amplification of multiple targets in a single PCR experiment.
• In a multiplexing assay, more than one target sequence can be
amplified by using multiple primer pairs in a reaction mixture.
• As an extension to the practical use of PCR, this technique has
the potential to produce considerable savings in time and
effort within the laboratory without compromising on the
utility of the experiment.
• Annealing temperatures for each of the primer sets must be
optimized to work correctly within a single reaction, and
amplicon sizes, i.e., their base pair length, should be different
enough to form distinct bands when visualized by gel
electrophoresis.
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32. TYPES OF MULTIPLEX PCR
1. Single template PCR reaction; this technique uses a
single template which can be a genomic DNA along with
several pairs of forward and reverse primers to amplify
specific regions within a template
2. Multiple template PCR reaction; this technique uses
multiple templates and several primer sets in the same
reaction tube. Presence of multiple primer may lead to
cross hybridization with each other and the possibility of
mis-priming with other templates.
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33. Primer Design Parameters for Multiplex
PCR
• Design of specific primer sets is essential for a successful
multiplex reaction. The important primer design
considerations described below are a key to specific
amplification with high yield.
• Primer Length: Multiplex PCR assays involve designing of
large number of primers, hence it is required that the designed
primer should be of appropriate length. Usually, primers of
short length, in the range of 18-22 bases are used.
• Melting Temperature: Primers with similar Tm, preferably
between 55°C-60°C are used. For sequences with high GC
content, primers with a higher Tm (preferably 75°C-80°C) are
recommended. A Tm variation of between 3°-5° C is acceptable
for primers used in a pool.
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34. • Specificity: It is important to consider the specificity of designed
primers to the target sequences, while preparing a multiplex
assay, especially since competition exists when multiple target
sequences are in a single reaction vessel.
• Avoid Primer Dimer Formation: The designed primers should
be checked for formation of primer dimers, with all the primers
present in the reaction mixture. Dimerization leads to unspecific
amplification.
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36. Advantages of Multiplex PCR
1. Internal Controls: Potential problems in a simple PCR include
false negatives due to reaction failure or false positives due to
contamination. False negatives are often revealed in
multiplex assays because each amplicon provides an internal
control for the other amplified fragments.
2. Efficiency: The expense of reagents and preparation time is
less in multiplex PCR than in systems where several tubes of
uniplex PCRs are used. A multiplex reaction is ideal for
conserving costly polymerase and templates in short supply.
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37. 3. Indication of Template Quality: The quality of the
template may be determined more effectively in
multiplex than in a simple PCR reaction.
4. Indication of Template Quantity: The exponential
amplification and internal standards of multiplex PCR
can be used to assess the amount of a particular template
in a sample. To quantitate templates accurately by
multiplex PCR, the amount of reference template, the
number of reaction cycles, and the minimum inhibition
of the theoretical doubling of product for each cycle must
be accounted.
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38. USES OF MULTIPLEX PCR
• Its has been found useful in Pathogen Identification,
High Throughput SNP Genotyping, Mutation Analysis,
Gene Deletion Analysis, Template Quantification,
Linkage Analysis, RNA Detection and Forensic Studies.
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39. METHYLATION-SPECIFIC PCR
(MSP)
• Methylation-specific PCR (MSP) is used to identify
patterns of DNA methylation at cytosine-guanine (CpG)
islands in genomic DNA .
• Target DNA is first treated with sodium bisulphite, which
converts unmethylated cytosine bases to uracil, which is
complementary to adenosine in PCR primers.
• Two amplifications are then carried out on the
bisulphite-treated DNA: One primer set anneals to DNA
with cytosines (corresponding to methylated cytosine),
and the other set anneals to DNA with uracil
(corresponding to unmethylated cytosine).
• MSP used in Q-PCR provides quantitative information
about the methylation state of a given CpG island.
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40. • Treatment of DNA with bisulphite
converts cytosine residues to uracil,
but leaves 5-methylcytosine residues
unaffected.
• Thus, bisulphite treatment
introduces specific changes in the
DNA sequence that depend on the
methylation status of individual
cytosine residues, yielding single-
nucleotide resolution information
about the methylation status of a
segment of DNA.
• The objective of this analysis is
therefore reduced to differentiating
between single nucleotide
polymorphisms (cytosines and
thymidine) resulting from bisulphite
conversion.
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41. • The MethyLight method is based on MSP, but provides a
quantitative analysis using real-time PCR.
• Methylated-specific primers are used, and a methylated-
specific fluorescence reporter probe is also used that
anneals to the amplified region.
• In alternative fashion, the primers or probe can be designed
without methylation specificity if discrimination is needed
between the CpG pairs within the involved sequences.
• Quantitation is made in reference to a methylated
reference DNA. A modification to this protocol to increase
the specificity of the PCR for successfully bisulphite-
converted DNA (ConLight-MSP) uses an additional probe
to bisulphite-unconverted DNA to quantify this non-
specific amplification.
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42. Methylation-specific PCR flow
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43. NESTED PCR
• This PCR increases the specificity of DNA amplification,
by reducing background due to non-specific
amplification of DNA.
• Two sets (instead of one pair) of primers are used in two
successive PCRs.
• In the first reaction, on pair of primers “outer pair” is
used to generate DNA products, which besides the
intended target, may still consist of non-specifically
amplified DNA fragments.
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44. • The product(s) are then used in a second PCR after the
reaction is diluted with a set of second set “nested or
internal” primers whose binding sites are completely or
partially different from and located 3' of each of the
primers used in the first reaction.
• The specificity of PCR is determined by the specificity of
the PCR primers.
• For example, if your primers bind to more than one locus
(e.g. paralog or common domain), then more than one
segment of DNA will be amplified. To control for these
possibilities, investigators often employ nested primers to
ensure specificity.
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45. • Nested PCR means that two pairs of PCR primers were
used for a single locus.
• The first pair amplified the locus as seen in any PCR
experiment.
• The second pair of primers (nested primers) bind
within the first PCR product and produce a second PCR
product that will be shorter than the first one.
• The logic behind this strategy is that if the wrong locus
were amplified by mistake, the probability is very low
that it would also be amplified a second time by a second
pair of primers.
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46. NESTED PCR SCHEME
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47. . Nested PCR strategy. Segment of DNA with dots representing
nondiscript DNA sequence of unspecified length. The double lines
represent a large distance between the portion of DNA illustrated in
this figure. The portions of DNA shown with four bases in a row
represent PCR primer binding sites, though real primers would be
longer.
The first pair of PCR primers (blue with arrows) bind to the outer
pair of primer binding sites and amplify all the DNA in between
these two sites
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48. PCR product after the first round of amplificaiton. Notice
that the bases outside the PCR primer pair are not present
in the product.
Second pair of nested primers (red with arrows) bind to
the first PCR product. The binding sites for the second pair
of primers are a few bases "internal" to the first primer
binding sites.
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49. Final PCR product after second round of PCR. The
length of the product is defined by the location of
the internal primer binding sites.
USES OF NESTED PCR
When a complete genome sequence is known, it is easier to be
sure you will not amplify the wrong locus but since very few of
the world's genomes have been sequenced completely, nested
primers will continue to be an important control for many
experiments.
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50. QUANTITATIVE PCR (Q-PCR)
• Used to measure the quantity of a PCR product (commonly in real-
time).
• It quantitatively measures starting amounts of DNA, cDNA or RNA.
• Q-PCR is commonly used to determine whether a DNA sequence is
present in a sample and the number of its copies in the sample.
• Quantitative real-time PCR has a very high degree of precision.
• QRT-PCR methods use fluorescent dyes, such as Sybr Green,
EvaGreen or fluorophore-containing DNA probes, such as TaqMan,
to measure the amount of amplified product in real time.
• It is also sometimes abbreviated to RT-PCR (Real Time PCR) or RQ-
PCR. QRT-PCR or RTQ-PCR are more appropriate contractions, since
RT-PCR commonly refers to reverse transcription PCR , often used in
conjunction with Q-PCR.
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52. SYBR Green I
hn
ssDNA -- unbound dye
minimal fluorescence
hn
dsDNA -- bound dye >100
fold increase fluorescence
TaqM an -- Hydrolysis Probe
M onitor acceptor fluorescence
hn
Hybridization probes
FRET
hn
donor acceptor
hn
fluor quencher
hn
Extension continues
Figure 2. Figure X. Schematic of SYBR Green I, TaqMan, and hybridization probe
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53. REVERSE TRANSCRIPTION PCR (RT-PCR)
• A PCR designed for amplifying DNA from RNA.
• Reverse transcriptase reverse transcribes RNA into
cDNA, which is then amplified by PCR.
• RT-PCR is widely used in expression profiling, to
determine the expression of a gene or to identify the
sequence of an RNA transcript, including transcription
start and termination sites.
• If the genomic DNA sequence of a gene is known, RT-
PCR can be used to map the location of exons and
introns in the gene.
• The 5' end of a gene (corresponding to the transcription
start site) is typically identified by RACE-PCR (Rapid
Amplification of cDNA Ends)
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54. Reverse Transcription PCR flow 548/14/2014

55. With PCR, it is now possible to amplify and examine minute
quantities of rare genetic material: the limit of this exploration
being the single cell.
Single cell PCR has applications in many areas, and has great
application especially in the field of prenatal diagnostics.
In prenatal diagnosis, single cell PCR has made possible
preimplantation genetic analysis and the use of fetal cells enriched
from the blood of pregnant women for the assessment of single-
gene Mendelian disorders.
Single-cell PCR has not only proven its usefulness in diagnostics, but
also lately has been very useful to basic scientists investigating
immunological, neurological and developmental problems.
Single Cell PCR
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56. THERMAL ASYMMETRIC INTERLACED
PCR (TAIL-PCR)
• This reaction is applied in the isolation of an unknown
sequence flanking a known sequence. Within the known
sequence, TAIL-PCR uses a nested pair of primers with
differing annealing temperatures; a degenerate primer is
used to amplify in the other direction from the unknown
sequence .
• Uses: 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 while the recover insert ends can also be used
for chromosome walking and mapping.
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57. • Nested, insertion-specific primers are used together with
arbitrary degenerate primers (AD primers), which are
designed to differ in their annealing temperatures.
• Alternating cycles of high and low annealing temperature
yield specific products bordered by an insertion-specific
primer on one side and an AD primer on the other.
• Further specificity is obtained through subsequent rounds
of TAIL-PCR, using nested insertion-specific primers.
• The increasing availability of whole genome sequences
renders TAIL-PCR an attractive tool to easily identify
insertion sites in large genome tagging populations through
the direct sequencing of TAIL-PCR products.
• For large-scale functional genomics approaches, it is
desirable to obtain flanking sequences for each individual
in the population in a fast and cost-effective manner.
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61. • Alu PCR
The pcr is performed using Alu primers designed to have
recognition sequence of Alu restriction enzyme. Used as a method
of obtaining a fingerprint of bands from an uncharacterized human
DNA.
• LONG PCR
Long PCR is a PCR is which extended or longer than standard PCR,
meaning over 5 kilobases (frequently over 10 kb). Long PCR is
usually only useful if it is accurate. Thus, special mixtures of
proficient polymerases along with accurate polymerases such as Pfu
are often mixed together.
Applications of Long PCR Long PCR is often used to clone larger
genes or large segments of DNA which standard PCR cannot.
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62. • AP-PCR
Arbitrarily Primed PCR (AP-PCR) or
Random Amplified Polymorphic DNA (RAPD) are methods of
creating genomic fingerprints from species of which little is
known about target sequence to be amplified.
• COLONY PCR
The screening of bacterial (E.Coli) or yeast clones for correct
ligation or plasmid products . Selected colonies of bacteria or
yeast are picked with a sterile toothpick or pipette tip from a
growth (agarose) plate.
This is then inserted into the PCR master mix or pre-inserted
into autoclaved water. PCR is then conducted to determine if
the colony contains the DNA fragment or plasmid of interest.
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63. TAP-PCR
AP PCR run at three different annealing
temperature
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64. LAMP assay: (Loop-mediated isothermal
amplification)
• It is a Modified type of the PCR using 3-6 primers sets
one of them is loop like primer.
• This test use Bst-polymerase enzyme
(Bacillus stearothermophilus DNA Polymerase).
• Using only two temperatures (63°C and 85°C for one
hour), may be carry out in water bath.
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65. THE DIGITAL PCR
• The Digital polymerase chain reaction simultaneously amplifies thousands
of samples, each in a separate droplet within an emulsion .
OVERLAP-EXTENSION PCR
• A genetic engineering technique allowing the construction of a DNA
sequence with an alteration inserted beyond the limit of the longest
practical primer length .
SOLID PHASE PCR
• Encompasses multiple meanings, including Colony Amplification (where
PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (primers
are covalently linked to a solid-support surface), conventional Solid Phase
PCR (where Asymmetric PCR is applied in the presence of solid support
bearing primer with sequence matching one of the aqueous primers) and
Enhanced Solid Phase PCR (where conventional Solid Phase PCR can be
improved by employing high Tm and nested solid support primer with
optional application of a thermal 'step' to favour solid support priming)
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66. • Box PCR
Box elements are repetitive sequence elements in
bacterial genome such as Streptococcus genome. Single
primer targeting to the repeats can be used to fingerprint
bacterial species.
• Competitive PCR(cPCR)
this is a method used for quantifying DNA using real
time PCR. A competitor internal standard is co amplified
with the target DNA and the target is quantified from the
melting curves of the target and its competitor.
• Consensus PCR
This PCR is carried out by using flanking primers to
amplify repeat regions from a no. of species. In this case
degenerate/consensus primers can be used for amplifying
the flanking sequences.
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67. Degenerate PCR
•In this instead of using specific PCR primers for a given sequence,
mixed PCR primers will be used.
•That is “wobbles” are inserted into the primers in case if the exact
sequence of gene is not known, so that there will be more than one
possibility for exact amplifications.
•Degenerate PCR has proven to be a powerful tool to find ‘new’
gene or gene families. By aligning the sequences from a no. of
related proteins the conserved and variables part can be
determined.
•Based on this information one can use conserved protein motifs
for starting points for designing degenerate PCR primers.
Degenerate oligonucleotide-primed PCR(DOR PCR)
• PCR amplification of limited sample by using degenerate PCR
primers is called DOR PCR.
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68. • Differential display PCR(DD PCR)
It is used for cloning purpose; it combines the comparative
analysis of several samples with the sensitivity of PCR.
• Forensic PCR
The VNTR locus is PCR amplified to compare DNA samples from
different sources.
• Hairpin PCR
A method for error free DNA amplification for mutation
detection. It first converts a DNA sequence to a hairpin. True
mutations will maintain hairpin structure during amplification
while PCR errors will disrupt the hairpin structure.
• PCR ELISA
The PCR products are labeled(digoxigenin) during amplification.
A capture probe specific to PCR amplicon is used to immobilize the
amplicon to immune-well plate. ELISA is then used against the
label(anti-digoxigenin) to quantitate PCR products.
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69. TOUCHDOWN PCR (STEP-DOWN PCR)
• A variant of PCR that aims to reduce nonspecific background by
gradually lowering the annealing temperature as PCR cycling
progresses. The annealing temperature at the initial cycles is
usually a few degrees (3-5°C) above the Tm of the primers used,
while at the later cycles, it is a few degrees (3-5°C) below the
primer Tm. The higher temperatures give greater specificity for
primer binding, and the lower temperatures permit more efficient
amplification from the specific products formed during the initial
cycles .
MINIPRIMER PCR
• This reaction uses a thermostable polymerase (S-Tbr) that can
extend from short primers ("smalligos") as short as 9 or 10
nucleotides. This method permits PCR targeting to smaller
primer binding regions, and is used to amplify conserved DNA
sequences, such as the 16S (or eukaryotic 18S) rRNA gene.
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70. • RACE PCR
Rapid Amplification of cDNA Ends PCR. This technique is
used to obtain 3’end of a cDNA; it requires some sequence
information internal to mRNA under study. The sequence
information obtained from this technique can be utilized to
obtain full length cDNA clones using the 5’RACE technique.
• Rep-PCR
Is used for Genomic Fingerprinting of plant-associated
bacteria and computer-assisted plant analyses. The genomic
fingerprinting method employed is based on the use of DNA
primers corresponding to naturally occurring interspersed
repetitive elements in bacteria such as REP,ERIC and BOX
elements.
• Vectorette-PCR
This method enables the amplification of specific DNA
fragments in situation where sequence of only one primer is
known. Thus it extends the application of PCR to the stretches
of DNA where the sequence information is only available at one
end.
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72. UNIVERSAL FAST WALKING PCR
• Used for genome walking and genetic fingerprinting using a more specific
'two-sided' PCR than conventional 'one-sided' approaches (using only one
gene-specific primer and one general primer - which can lead to artefactual
'noise') by virtue of a mechanism involving lariat structure formation.
Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested
PCR for rapid amplification of genomic DNA ends), 5'RACE LaNe and 3'RACE
LaNe .
VARIABLE NUMBER OF TANDEM REPEATS (VNTR) PCR
• This method targets areas of the genome that exhibit length variation. The
analysis of the genotypes of the sample usually involves sizing of the
amplification products by gel electrophoresis. Analysis of smaller VNTR
segments known as Short Tandem Repeats (or STRs) is the basis for DNA
Fingerprinting databases such as CODIS .
INTERSEQUENCE-SPECIFIC PCR (OR ISSR-PCR)
• This is a method for DNA fingerprinting that uses primers selected from
segments repeated throughout a genome to produce a unique fingerprint of
amplified product lengths. The use of primers from a commonly repeated
segment is called Alu-PCR, and can help amplify sequences adjacent (or
between) these repeats. 728/14/2014

73. REFERENCES
8/14/2014 73
• Introduction to Plant Biotechnology By- H.S. Chawala
• Yasumasa Kimura et al. Optimization of turn-back
primers in isothermal amplification
(http://www.ncbi.nlm.nih.gov/pmc/articles/PMC308
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• R. Manojkumar and Mrudula Varanat(2006) Polymerase
Chain Reaction: Types and Its Application in theField of
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