Nucleic acids amplification

Dr.Salma Mahmoud
 Nucleic acid amplification is a valuable molecular tool not only in
basic research but also in application oriented fields, such as clinical
medicine development, infectious diseases diagnosis, gene cloning
and industrial quality control.
 PCR was the first nucleic acid amplification method.
 It is the preferred method for application oriented fields involving
nucleic acid amplification for its simplicity, easier methodology,
extensively validated standard operating procedure and availability
of reagents and equipments.
 However, PCR has a good no of limitations, including high cost of
equipment, contamination chances, sensitivity to certain classes of
contaminants and inhibitors, requirement of thermal cycling, etc
 Most of the alternative methods are isothermal obviating the need
for thermal cyclers. (isothermal :using a temperature that does not
change)
 Though principles of most of the alternate methods are relatively
complex than that of PCR, they offer better applicability and
sensitivity in cases where PCR has limitations.
 Amplification methods
 Several alternative amplification methods have been developed
 These methods increase sensitivity of test system by making more
copies of target, or probe, or attaching more signal producing
molecules onto target.
 Categories:
1/ Target amplification methods:
 PCR
 RT-PCR
 qPCR
 Transcription based amplification
 Transcription-mediated amplification (TMA; GenProbe)
 Nucleic acid sequence-based amplification (NASBA)
2/ Probe amplification methods:
 Ligase chain reaction (LCR)
 Strand displacement amplification (SDA)
 QB replicase
3/Signal amplification methods:
 Branched chain DNA (bDNA) amplification
 Hybrid capture assay (HCA)
 Cleavage based amplification
 None of these techniques have achieved the same widespread research
application as PCR
 Most likely due to the simplicity and cost-effectiveness of PCR.
 However, some of these techniques have been incorporated into clinical
diagnostic assays
 SDA is a platform technology used
for Mycobacteria and Chlamydia detection
 NASBA is used for HIV-1, CMV and Enteroviruses
 TMA is used for the detection of Mycobacteria, Neisseria and Chlamydia.
 Conventional amplification techniques continue to play an integral
role in characterizing and genotyping parasites for medical,
environmental and epidemiological investigations.
 Techniques such as amplified restriction fragment length
polymorphism (AFLP), PCR-restriction fragment length
polymorphism (PCR-RFLP) and random amplified polymorphic
DNA (RAPD) are currently employed to answer specific biological
and evolutionary questions
 The greatest recent advancement in amplification technology has
been the development of systems that allow monitoring of
amplification in real-time.
 Target amplification methods
1/Polymerase Chain Reaction
 The PCR is a method for amplifying a DNA sequence region
located between two primers.

 Amplification is specific and highly sensitive, allowing a

target sequence to be specifically amplified starting from a
complex mixture of DNA
 Often the DNA sample is available in very limited quantities
(for example, crime scene evidence or very small tissue
samples from biopsies) or in an ancient condition that
resulted in the DNA being partially degraded (in museum
specimens, dried specimens, and fossils).
 To circumvent the problem of having small amounts of tissue
or nucleic acids, Kary Mullis, a biochemist devised the
polymerase chain reaction (PCR) in 1983, for which he won
the 1993 Nobel Prize in Chemistry.
 PCR can be used to amplify whatever DNA is present in a
sample, however small in quantity or poor in quality.
 The only requirement is that a short sequence of nucleotides
on either side of the sequence of interest be known.
 These short sequences can be generated from known
nucleotide sequences or from deduced, reverse-translated
amino acid sequences.
 This sequence information is needed to construct primers on
either side of the sequence of interest.
 The primers are usually 15–25 nt in length.
 As with Southern and Northern hybridizations, the primers
do not need to exactly base-pair with the target because the
annealing conditions can be adjusted.
 In the PCR technique, the template, primers, DNA
polymerase, and dNTPs are combined.
 The following procedure then is followed:
 1.The mixture is heated (for example, 92°C for 30 sec) to
denature the DNA.(Denaturation)
 2. The temperature is then lowered (for example, 56°C
 for 30 sec) to allow the primers to anneal to their
complementary target sequences.(Annealing)
 Adjusting this temperature allows for more- or less-perfect
base pairing between the primers and the target sequences.
 3. The temperature is then increased (for example, 72°C) for
extension of the DNA strand. (Extension)
 The length of time for DNA extension (replication) depends
on the predicted length of the amplified product.
 Then, a new cycle of denaturing, annealing, and DNA
extension is initiated.
 By the end of Cycle 3, we have generated the desired product
that begins and ends with the oligonucleotide sequences on
both strands.
 This DNA product is then amplified exponentially through
subsequent cycles.
 About 20 PCR cycles produce a million copies of DNA; 30
cycles make a billion copies.
 The various stages in the cycle can be controlled by
temperature because denaturation, primer annealing, and
DNA extension all require different temperatures.
 In the original description of the technique, DNA polymerase
had to be added just before each DNA extension step because
the denaturing temperature also inactivated DNA
polymerase.
 The technique became widely accepted when a DNA
polymerase from a hot springs bacterium, Thermus aquaticus,
was identified that could withstand the denaturing
temperatures.
 Using this polymerase, no additional enzyme had to be added
to the reaction mixture after each cycle
 Instead, the cycling could be continued without interruption
in PCR machines, which could be programmed to
automatically change temperatures after a specified length of
time.
 Furthermore, the ability of these machines to handle
hundreds of samples simultaneously has dramatically
automated the isolation of DNA sequences without the need
for making and screening genomic DNA and cDNA libraries.
2/Reverse Transcription PCR (RT-PCR)

 1. RNA template is converted to a DNA copy (cDNA) by RNA-

dependent DNA polymerase; also known as reverse transcriptase.

 2. cDNA product then serves as template to make millions of copies

of target RNA sequence
 3/Transcription-Based Amplification Systems

 RNA is the target and primary product.

 Reactions are isothermal.
 Each cycle of the TAS is composed of two steps.
 The first is a cDNA synthesis step that produces one copy of a
double-stranded DNA template for each copy of RNA or DNA target
nucleic acid.
 During the course of this cDNA synthesis step, a sequence
recognized by a DNA-dependent RNA polymerase is inserted into
the cDNA copy of the target sequence to be amplified.
 The second step is the amplification of the target sequence by the
transcription of the cDNA template into multiple copies of RNA.
 Applications:
 Direct detection of RNA viruses and RNA from other infectious
agents, as well as transcribed gene sequences
 This procedure has been applied to the detection of human
immunodeficiency virus type 1 (HIV-1)-infected cells.
 Probe Amplification Methods

 Number of target sequences in sample is not changed.

 Synthetic primers/probes complementary to target nucleic acid are
amplified.
 1/Ligase chain reaction(LCR)

 The ligase chain reaction (LCR) is an amplification process that

differs from PCR in that it involves a thermostable ligase to join
two probes or other molecules together which can then be amplified
by standard PCR cycling
 c. Enzymes: DNA polymerase synthesizes cDNA by extending
primers, and DNA ligase seals gap between adjacent primers.
 d. Thermocycler needed
 e. Applications: Detect point mutations in known genes (e.g., beta-
globin gene for detection of sickle cell disease)
 The method of DNA amplification is similar to PCR; however, LCR
amplifies the probe molecule rather than producing amplicon
through polymerization of nucleotides.
 LCR uses both a DNA polymerase enzyme and a DNA ligase
enzyme to drive the reaction.
 LCR uses two complementary pairs of oligonucleotides(four
oligonucleotide primers) that hybridize in close proximity on the
target fragment .
 Only when the oligonucleotides correctly hybridize to the target
sequence, the remaining nick between the oligonucleotides is
ligated by a DNA ligase and a fragment equating to the total
sequence of both oligonucleotides is generated
 Once the probes have been ligated, the ligation product can serve as
a template for annealing and future ligation.
 Like PCR, LCR requires a thermal cycler to drive the reaction and
each cycle results in a doubling of the target nucleic acid molecule.
 The detection of LCR products can be performed by
electrophoresis or by an enzyme-linked immunosorbent assay
(ELISA) like microplate procedure or real-time detection
 Steps:
 1/Heat denaturation
 2/Annealing of four probes
 3/Gap filling with thermostable polymerase
 4/Ligation with thermostable ligase.
 2/Strand displacement amplification(SDA)

 a. Major amplification products are the probes/primers.

 Used 2 special primers
 b. Denaturation, then isothermal two-stage process of :
 1/target generation followed by 2/exponential amplification
phase
 c. First stage involves target generation;
 Goal is to generate a new version of the DNA target we want to
amplify.
 Amplification of only one strand
 The amplification primer contain special restriction enzyme site
specific for only one region in template DNA
 Upstream to this there is a second primer(Bumper) binds
 Extension by DNA polymerase take place starting from the
amplification primer

 primer and probe bind close to each other. Probes have recognition
site for a restriction enzyme (RE).
 1) As the outer primers are extended, they displace the probes,
which are extended.
 2) A second set of complementary primers then binds to displaced
probes, and DNA polymerase extends the complementary primers,
producing a double-stranded version of the probes.
 3) The probes are the target DNA for the next stage of the process.
 d. Second stage: Exponential probe/target amplification phase.
 1) When RE is added to ds probe DNA, only one strand of the probe
will be cut, leaving a nick in the DNA that was extended.
 The opposite strand is simultaneously displaced.
 The displaced strand is copied by the primers.
 2) The process takes place at 52°C without temperature cycling and
produces millions of copies of the initial sequence.
 e. Addition of fluorogenic probe yields a fluorescent signal directly
proportional to the amount of amplified probe/target.
 f. Application: SDA is the basis for the BD ProbeTec®ET for
Mycobacterium tuberculosis, Chlamydia trachomastis, and
Neisseria gonorrhoeae.
 3/QB replicase

 QB replicase is an RNA-dependent RNA polymerase from bacteriophage

QB.
 Bacteriophage Qbeta, commonly referred to as Qbeta or Qβ, is a species
consisting of several strains of positive-strand RNA virus which infects
bacteria that have F-pili, most commonly Escherichia coli.
 The target can be either denatured DNA or RNA.
 a. Probe-bound template is amplified by mixing with QB replicase, which
can generate a billion RNA molecules/probes in less than 15 minutes.
 b. Used for identification of infectious agents
 Signal Amplification Methods

 1. bDNA amplification, HCA, and cleavage-based amplification

 2. In signal amplification systems, the number of target sequences
does not change; instead, large amounts of signal are bound to the
target sequences present in the sample, making detection more
sensitive.
 These systems carry less risk of target contamination.
 1/ Branched chain DNA (bDNA)

 3. b DNA is frequently used for quantification of target sequences in

clinical samples, especially viral load determinations.
 Several different short single-stranded DNA molecules
(oligonucleotides) are used in a branched DNA-assay.
 The capture and capture-extender oligonucleotide bind to the target
nucleic acid and immobilize it on a solid support.
 Procedure: A branched DNA assay begins with a dish or some
other solid support (e.g., a plastic dipstick).
 The dish is peppered with small, single stranded DNA molecules
(or chains) that stick out into the solution. These are known as
capture probe DNA molecules.
 Next, an extender DNA molecule is added.
 Each extender has two domains; one that hybridizes to the capture
DNA molecule and one that sticks out above the surface.
 Once the capture and extender molecules are in place and they have
hybridized, the sample can be added.
 Target molecules in the sample will bind to the extender molecule.
This results in a base peppered with capture probes, which are
hybridized to extender probes, which in turn are hybridized to target
molecules.
 At this point, signal amplification takes place.
 A label extender DNA molecule is added that has two domains
(similar to the first extender).
 The label extender hybridizes to the target and to a pre-amplified
molecule.
 The preamplifier molecule has two domains. First, it binds to the
label extender and second, it binds to the amplifier molecule.
 An example amplifier molecule is an oligonucleotide chain bound to
the enzyme alkaline phosphatase.
 2/ Hybrid capture assay (HCA)

 4. HCA is marketed by Digene Diagnostics for detection of human

papillomavirus, hepatitis B virus, and cytomegalovirus.
 a. Target DNA is released from cells and binds to ssRNA probes to
form DNA/RNA hybrid molecules.
 b. DNA/RNA hybrid forms unique structure, which can be bound
by antibodies to surface of microtiter well.
 c. Captured hybrids are detected by binding alkaline
phosphatase conjugated anti-DNA/RNA hybrid antibodies in a
typical "sandwich“ assay.
 d. Substrate is added and signal is measured.
 3/Cleavage-based amplification

 Based on activity of cleavase enzyme used in the Invader® assay

(Third Wave Technologies, Inc.)
 a. Detects target nucleic acid by a series of probes that bind to the
target and overlap
 b. Cleavase recognizes overlapping sequence of DNA and cuts it.
 c. During isothermal incubation, if probe and test sequences are
complementary, two enzymatic cleavage reactions occur, resulting
in a fluorescent signal.
 d. Applications: Used in genetics, hemostasis (e.g., factor V Leiden
mutation detection), and infectious disease