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Target Amplification Methods Students

Molecular techniques are increasingly used in disease diagnosis and evaluation. Positional cloning can locate disease-causing genes without knowing the protein product. Molecular analysis of cancer mutations may provide prognostic information and guide therapy selection. Gene therapy also uses detailed understanding of genetic diseases and cancer. Real-time PCR and other amplification methods like PCR, RT-PCR, and isothermal techniques exponentially increase target sequences for sensitive disease detection.

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Courtny Lenz Maygay Gapa
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57 views

Target Amplification Methods Students

Molecular techniques are increasingly used in disease diagnosis and evaluation. Positional cloning can locate disease-causing genes without knowing the protein product. Molecular analysis of cancer mutations may provide prognostic information and guide therapy selection. Gene therapy also uses detailed understanding of genetic diseases and cancer. Real-time PCR and other amplification methods like PCR, RT-PCR, and isothermal techniques exponentially increase target sequences for sensitive disease detection.

Uploaded by

Courtny Lenz Maygay Gapa
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
Available Formats
Download as PDF, TXT or read online on Scribd
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Relationship to Laboratory Evaluation of Disease

MOLECULAR DIAGNOSIS
Increasingly, molecular technology is being integrated into the mainstream of laboratory
testing.

One of the most important consequences of the revolution in molecular genetics has
been the ability to locate a gene responsible for a disease without knowing the protein
product.

Positional cloning, this process uses genomic mapping and linkage analysis of families
that carry and express the disease to locate markers closer and closer to the disease
gene until it is ultimately located.
Beyond Diagnosis

Molecular analysis of the somatic mutations in cancer has the potential to provide prognostic
information, guide the selection of optimal therapy, and monitor response to therapy.

The implications of using molecular markers to detect neoplastic cells in the absence of clinical or
morphologic evidence of disease, known as minimal residual disease, are now being explored in a
variety of malignancy types

Gene therapy for inherited genetic diseases as well as cancer is being introduced based on a detailed
under- standing of the underlying disease.
TARGET
AMPLIFICATION
METHODS
POLYMERASE CHAIN
REACTION
• PCR is a simple in vitro chemical reaction that permits the
synthesis of essentially limitless quantities of a targeted nucleic
acid sequence.
• Accomplished through the action of a deoxyribonucleic acid (DNA)
polymerase that, under the right conditions, can copy a strand of
DNA.
• A PCR consists of target DNA, a molar excess of two
oligonucleotide primers, a heat-stable DNA polymerase, an
equimolar mixture of deoxyribonucleotide triphosphates (dATP,
dCTP, dGTP, and dTTP), MgCl2, KCl, and a Tris-HCl buffer.
PCR
PROCESS
Reverse-Transcriptase
Polymerase Chain
Reaction
• PCR, as it was originally described,
was a technique for DNA
amplification. Reverse-
transcriptase PCR (RT-PCR) was
developed to amplify ribonucleic
acid (RNA) targets.
NESTED POLYMERASE
CHAIN REACTION
• Nested PCR was developed to increase both the
sensitivity and specificity of PCR.
• It employs two pairs of amplification primers and two
rounds of PCR.
• The products of the first round of amplification are
then subjected to the second round of amplification
using the second set of primers, which anneal to a
sequence internal to the sequence amplified by the first
primer set.
• The increased sensitivity arises from the high total
cycle number, and the increased specificity arises from
the annealing of the second primer set to sequences
found only in the first-round products, thus verifying
the identity of the first-round product.
• Major disadvantage: high risk contamination
Real-Time (Homogeneous, Kinetic) Polymerase
Chain Reaction

• Real-time PCR describes methods by which the target


amplification and detection steps occur
simultaneously in the same tube (homogeneous).
• These methods require special thermal cyclers with
precision optics that can monitor the fluorescence
emission from the sample wells.
• The computer software supporting the thermocycler
monitors the data throughout the PCR at every cycle
(kinetic) and generates an amplification plot for each
reaction.
• The PCR cycle at which the fluorescence passes the
threshold is defined as the cycle threshold (CT).
• There is an inverse linear relationship between the
log of the initial target concentration and the CT.
• SYBR Green I is one such dye used in this
application.
RT-PCR (cont)
• The specificity of real-time PCR can also be increased
by including hybridization probes in the reaction
mixture.
• These probes are labeled with fluorescent dyes or
with a combination of fluorescent and quencher dyes.
• In the 5′ nucleases (TaqMan) PCR assay, the 5′ to 3′
exonuclease activity of Taq DNA polymerase is used
to cleave a non-extendable hybridization probe
during the primer extension phase of PCR (Holland et
al, 1991).
• Fluorescence resonance energy transfer (FRET)
probes can also be used to detect PCR product as it
is generated (Lay & Wittwer, 1997).
• This method requires two specially designed
sequence-specific oligonucleotide probes.
TRANSCRIPTION-BASED
AMPLIFICATION
• Transcription-based amplification
includes transcription-mediated
amplification (TMA) and nucleic acid
sequence-based amplification
(NASBA).
• These techniques essentially
recapitulate retroviral replication in
vitro, converting RNA into DNA and
then using the DNA as a template for
transcription of multiple copies of RNA.
• NASBA and TMA have been used
successfully in a wide variety of clinical
qualitative and quantitative
applications, primarily in infectious
diseases.
STRAND-DISPLACEMENT
AMPLIFICATION
• Strand-displacement amplification (SDA) is an
isothermal template amplification technique that can be
used to detect trace amounts of DNA or RNA of a
particular sequence.
• In its current iteration, SDA occurs in two discrete
phases: target generation and exponential target.
• In the target generation phase, a dsDNA target is
denatured and hybridized to two different primer pairs,
designated as bumper and amplification primers.
• The amplification primers include the single-
stranded restriction endonuclease enzyme
sequence for BsoB1 located at the 5′ end of the
target binding sequence.
• The bumper primers are shorter and anneal to the
target DNA just upstream of the region to be
amplified.
• FDA-cleared assays for detection of N. gonor- rhoeae,
Chlamydia trachomatis, Herpes simplex virus types 1
and 2, and T. vaginalis using SDA are available from BD
Diagnostics (Franklin Lakes, N.J.) on their ViperTM
system.
Loop-mediated amplification (LAMP) is an isothermal
LOOP-MEDIATED method that relies on autocycling strand displacement

AMPLIFICATION DNA synthesis by Bst DNA polymerase and a set of


four to six primers.

The products can be analyzed in real-time by


monitoring of the turbidity in the reaction tube
resulting from production of magnesium
pyrophosphate precipitate during the DNA
amplification.

Amplification products can also be visualized in


agarose gels after electrophoresis and staining with
ethidium bromide or SYBR Green.

The final products of the LAMP reaction are DNA


molecules with a cauliflower-like structure of multiple
loops consisting of repeats of the target sequence.
HELICASE-DEPENDENT
AMPLIFICATION
Helicase-dependent amplification (HDA) is an isothermal process developed by
BioHelix (Beverly, Mass.) that uses helicase to separate dsDNA and generate
single-stranded templates for primer hybridization and sub- sequent extension by
a DNA polymerase

In HDA, strands of dsDNA are separated by the DNA helicase and the ssDNA-
coated ssDNA-binding proteins.

HDA is compatible with multiple detection technologies, including qualitative and


quantitative fluorescent technologies and with instruments designed for real-time
PCR.
NICKING ENDONUCLEASE
AMPLIFICATION
Nicking endonuclease amplification is an isothermal process that begins with nicking of a double-
stranded DNA template by a nicking enzyme.

A DNA polymerase then binds at the nick sites and unravels the template. The T2 primer that includes
the nicking enzyme recognition site binds loosely to the template and is extended by polymerase.

A second T2 primer binds to the same target and is extended, displacing the first T2.

The nick sites within primers allow for the process to repeat for rapid amplification of products.

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