Chapter 6- Nucleic Acid Amplification

TARGET AMPLIFICATION

Involves making many copies of a specific DNA sequence

A. Polymerase Chain Reaction

Basic PCR Procedure- DNA replication in a cell requires an existing double-stranded DNA as the
template to give the order of the nucleotide bases; the deoxyribonucleotide bases in the form of
deoxynucleotide triphosphates (dNTPs)— dATP, dGTP, dCTP, dTTP; DNA polymerase to catalyze
the addition of nucleotides to the growing strand; and a primer (synthesized by primase in vivo)
to which DNA polymerase adds subsequent bases.

 PCR essentially duplicates the in vivo replication of DNA in vitro, using the same components to
replicate DNA, with the same end result: one copy of double-stranded DNA becoming two
copies.
 In less than 2 hours, PCR can produce millions of copies, collectively called amplicons
 The real advantage of PCR is the ability to amplify specific targets.
 PCR presents the opportunity to amplify and effectively clone the target sequences.
 components of the reaction—DNA template, short oligonucleotide primers, nucleotides,
polymerase, and buffers—are subjected to an amplification program, which consists of a specifi
ed number of cycles that are divided into steps during which the samples are held at particular
temperatures for designated times.
STEPS:
 The amplification program starts with one doublestranded DNA target. In the first step
(denaturation), the double-stranded DNA is denatured into two single strands. This is
accomplished by heating the sample at 94°C to 96°C for several seconds to several minutes,
depending on the template.
 The next step in the amplification program, most critical for the specificity of the PCR, is the
annealing step. This is the second step of the PCR cycle where the two oligonucleotide primers
that will prime the synthesis of DNA anneal (hybridize) to complementary sequences on the
template
- Annealing temperatures will range from 50°C to 70°C and are usually established empirically
 The third and last step of the PCR cycle is the primer-extension step. This is where DNA synthesis
occurs. In this step, the polymerase synthesizes a copy of the template DNA by adding
nucleotides to the hybridized primers. DNA polymerase replicates the template DNA by
simultaneously extending the primers on both strands of the template. This step occurs at the
optimal temperature of the enzyme, 68°C to 72°C.
Components of PCR

Other types/modifications of PCR

1. Touchdown PCR- is a modification of the PCR program used to enhance the
amplification of the desired PCR product. In this method, the PCR program begins with
annealing temperatures higher than the optimal target primer-binding temperature.
The annealing temperature is decreased by 1°C every cycle or every other cycle until the
optimal annealing temperature is reached.
 2. Multiplex PCR - More than one primer pair can be added to a PCR so that multiple
amplifi cations are primed simultaneously, resulting in the formation of multiple
products. Multiplex PCR is especially useful in typing or identifi cation analyses.
Individual organisms, from viruses to humans, can be identifi ed or typed by observing a
set of several PCR products at once.

3. Sequence-Specific PCR- The strict requirement for complementarity of the 3 ′ end of

primers can be used to identify single-base changes in the target DNA. By designing the
forward or reverse primer to end with a 3 ′ base complementary to the mutant
sequence, the presence of the base change is detected by successful amplification.

- Sequence-specific PCR is one of the methods of human leukocyte antigen allele analysis in
tissue typing.

4. Reverse Transcriptase PCR - If the starting material for a procedure is RNA, the RNA may
first be converted to double-stranded DNA, which is a better template for amplification
than single-stranded RNA.
 The conversion is accomplished through the action of reverse transcriptase (RT), an
enzyme isolated from RNA viruses. This enzyme first copies the RNA single strand
into an RNA:DNA hybrid and then uses a hairpin formation on the end of the newly
synthesized DNA strand to prime synthesis of the complementary DNA strand,
replacing the original RNA in the hybrid. The resulting double-stranded DNA is copy
or complementary DNA (cDNA).

5. Nested PCR - The increased sensitivity that PCR offers is very useful in clinical
applications because clinical specimens are often limited in quantity and quality.
 Nested PCR is a modification that increases the sensitivity and specificity of the
reaction.
 In nested PCR, two pairs of primers are used to amplify a single target in two
separate PCR runs. The second pair of primers, designed to bind slightly inside of the
binding sites of the first pair, will amplify the product of the first PCR in a second
round of amplification. The second amplification will specifically increase the
amount of the intended product.

6. Real-Time (Quantitative) PCR - A very useful modification of the PCR process is real-
time or quantitative PCR (qPCR).
 This method was first performed by adding ethidium bromide (EtBr) to a
standard PCR. Because EtBr intercalates into double-stranded DNA and
fluoresces, it tracks the accumulation of PCR products during the PCR in real
time, that is, as it is made.
 Using the fluorescent signal to detect the growing target copy number during
the amplification process, analysis in qPCR is performed in the exponential
phase of growth
  The PCR cycle at which sample fluorescence crosses the threshold is the
threshold cycle, or CT
 The first approach to qPCR utilized EtBr, which is specific to double-stranded
DNA. Dye is still used for routine qPCR, except that EtBr has been replaced by
SYBR green, another dye specific to double-stranded DNA.
 The advantage of SYBR green is its specificity and robust fluorescence
comparable to that of EtBr and its reduced toxicity

Molecular Beacons, measures the accumulation of product at the annealing step in the PCR cycle. The
signal from Molecular Beacons is detectable only when the probes are bound to the template before
displacement by the polymerase.

- Here the probe is chemically modified so that it is not degraded during the extension step.
- Molecular Beacons are designed with a target-specific binding sequence of approximately
25 bases fl anked by a short, approximately 5-base-long inverted repeat that will form a
stem and loop structure when the probe is not bound to the template.

Fluorescent resonance energy transfer (FRET), utilizes two specific probes, one with a 3 ′ fluorophore
(acceptor) and the other with a 5 ′ catalyst for the fluorescence (donor), that bind to adjacent targets.

Transcription-Based Amplification Systems

In a transcription-based amplification system (TAS), RNA is the usual target instead of DNA:

1. transcription-mediated amplification (TMA), 2. nucleic acid sequence–based amplification

(NASBA), and 3. self-sustaining sequence replication (3SR).

Genomic Amplification Methods

1. Whole-Genome Amplification WGA can be performed using degenerative primers that prime
random synthesis throughout the target genome or by nick translation.
- WGA has multiple applications, including comparative genomic arrays, detection of single-
nucleotide polymorphisms, and analysis of minimal starting material, such as DNA in plasma,
single-cell analysis, and ancient DNA samples.

2. Emulsion PCR- is designed to simultaneously amplify thousands of specific templates in a single

reaction, producing a set of specific products, or library.
- In this method, the ends of fragmented template DNA are ligated to universal primer
sequences and, along with the other components of the PCR reaction (buffer, polymerase
enzyme, forward and reverse primers, dNTPs), are added to an oil surfactant mixture (Span
80, Tween 80, Triton X-100, and mineral oil)
- With stirring, an emulsion is formed of 1 to 10 billion droplets, each of which can contain a
single template in a tiny volume of reaction buffer. When the emulsion is subjected to an
amplification program, the droplets will act as single reaction chambers producing many
copies of a specific sequence.
 3. Surface Amplification (Bridge PCR)-
Surface amplifi cation is an isothermal, genomic PCR method used in high-throughput
sequencing technologies. In this method, forward and reverse primers are immobilized on a
solid support in a flow cell
4. Abitrarily Primed PCR- also known as randomly amplified polymorphic DNA or random
amplification of polymorphic DNA (RAPD), short primers (10 to 15 bases) with random
sequences are used to amplify arbitrary regions in genomic DNA under low-stringency
conditions. With this method, PCR products are generated without knowing the sequence of the
target or targeting a specific gene
- Arbitrarily primed PCR has been used primarily in the epidemiological typing of
microorganisms.

PROBE AMPLIFICATION

In probe amplification procedures, the number of target nucleic acid sequences in a sample is not
changed. Rather, synthetic probes that are specifi c to the target sequences bind to the target where the
probes themselves are amplified.

1. Ligase Chain Reaction

-LCR was a method for amplifying synthetic primers/ probes complementary to target nucleic
acid
- the primers are bound immediately adjacent to each other. Instead of DNA polymerase
synthesizing complementary DNA by extending the primers as occurs in PCR, DNA ligase was
used in LCR to ligate the adjacent primers together.
- LCR required a thermal cycler to change the temperature to drive the different reactions. In
LCR the reaction was heated to denature the template.
- LCR was used to detect point mutations in a target sequence. EX. Sickle cell anemia

2. Strand Displacement Amplification

- Strand displacement amplification (SDA) is an isothermal amplification process; that is, after an
initial denaturation step, the reaction proceeds at one temperature

- In SDA the major amplification products are the probes. There are two stages to the SDA
process. The first stage of SDA is the denaturation of the double-stranded target and annealing
of primers and probes tailed with sequences including a restriction enzyme site. A second
reaction copies the probe, incorporating dATP α S and thereby inactivating the restriction site on
the copied strand. This species is the target for amplification in the second stage of the reaction.

- In the second phase of SDA, the target sequence is nicked by the restriction enzyme,
generating a substrate for the polymerase, which extends the nick, displacing the opposite
strand, and regenerates a new template for nicking. Copies of the displaced strands collect as
the reaction proceeds. The reaction cycles by the strands are nicking and extension, without
requirement for heat denaturation of the double-stranded DNA template.
3. Q β Replicase

- Q β replicase is another method for amplifying probes that have specifi city for a target sequence. The
method is named for the major enzyme that is used to amplify probe sequences. Q β replicase is an
RNA-dependent RNA polymerase from the bacteriophage Q β. The target nucleic acid in this assay can
be either DNA (which must first be denatured) or RNA.

The Q β replicase method proceeds through a series of binding and washing steps. Probe bound to the
purified template is then amplified by Q β replicase. The resulting RNA can be detected by fluorometry
using propidium iodide as a fluorescent label of the synthesized probe or by chromogenic methods.

SIGNAL AMPLIFICATION

In signal amplification procedures, there is no change in the number of target or probe sequences;
instead, large amounts of signal are bound to the target sequences that are present in the sample.
Signal amplification procedures are inherently better at quantifying the amount of target sequences
present in the clinical sample.

1. Branched DNA Amplification- a series of short oligomer probes is used to capture a single target
nucleic acid molecule.
- The target is captured or immobilized to a solid support by capture probes, after which
extender probes and blocking probes create a stable cruciform structure with the amplifiers.
Each amplifier has hybridization sites for 8 to 14 branches, which in turn bind substrate
molecules for alkaline phosphatase.
- Second-generation bDNA assays use extender probes that bind multiple amplifiers,
increasing the signal intensity and improving limits of detection.

2. Hybrid Capture Assays- were marketed primarily for the detection and molecular
characterization of human papillomavirus (HPV) in genitourinary specimens. 58,59 They are also
used for the detection of hepatitis B virus and CMV
- Hybrid capture starts with hybridization of the RNA probe to the denatured DNA target. The
RNA:DNA hybrid is then bound by hybrid-specific immobilized antibodies. A secondary
antibody bound to alkaline phosphatase generates signal in the presence of a
chemiluminescent substrate.
- The hybrid capture assay is considered a signal amplification assay because the amount of
target DNA is not amplified; rather, the DNA is isolated bound to RNA and is recognized by
multiple antibodies to the target/probe hybrid molecule

3. Cleavage-Based Amplification- detects target nucleic acids by using a series of overlapping

probes that bind to the target DNA.
- Cleavase is a bacterial enzyme that recognizes overlapping sequences of DNA and makes a
cut (cleaves) in the overlapping region.
- Invader assays exploit the substrate requirements of the enzyme. A cleavable substrate is
formed by the hybridization of two probes and template (top left). This structure will not
form if the probe does not match the template (top right). If the proper substrate is formed,
 the enzyme removes part of one probe, and that fragment forms another cleavable complex
with the fl uorescently labeled reporter probe (bottom left). Repeated binding and cleavage
amplify the signal. In a plate format, only those wells with a template complementary to the
test probe will produce fl uorescent signal

4. Cycling Probe
- In the cycling probe method of amplifi cation, target sequences are detected using a
synthetic probe consisting of sequences of DNA and RNA arranged in a DNA– RNA–DNA
sandwich sequence carrying a reporter dye at one end and a quencher dye at the other.
- Cycling probe produces fluorescence only when the RNA probe binds to the DNA template.
The RNA:DNA hybrid formed by the probe bound to the template is a substrate for RNase H,
which digests the RNA probe and releases the reporter dye (R) from the vicinity of the
quencher (Q).