Amplification of
Genomic DNA Fragments
OrR
Amplification
• To get particular DNA in large amount
• Fragment size shouldn’t be too long
• The nucleotide sequence at each end is
known.
How to get known sequence from both end of
particular genome fragment?
• Generally we do partially digestion of a
genome.
• Result fragments with known RE site in both
ends.
• Insert those in vector/plasmid particular
region.
• We know the sequence of the plasmid.
• Taking some part of the plasmid along with
restriction site as primers.
PCR reaction for amplification
Steps in PCR reaction:
Summary of PCR cycle:
The Power of Polymerase Chain Reaction
1. Number of Copies
exponential progression: 1, 2, 4, 8, 16, 32, 64, 128, 256, 512, 1024, and so forth,
doubling with each cycle of replicationNumber of Copies
starting with a single molecule, 25 rounds of DNA replication will result in 225 = 3.4 x
107 molecules.
2. Requires Trace Amounts of Template DNA
The major advantage of PCR amplification is that it requires only trace amounts of
template DNA.
amplification is usually reliable with as few as 10–100 template molecules, which
makes PCR amplification 10,000–100,000 times more sensitive than detection via
nucleic acid hybridization.
The Power of Polymerase Chain Reaction
3. Sensitivity
The exquisite sensitivity of PCR amplification has led to its use in DNA typing for
criminal cases in which a minuscule amount of biological material has been left
behind by the perpetrator (skin cells on a cigarette butt or hair-root cells on a single
hair can yield enough template DNA for amplification).
4. The Limitations of Polymerase Chain Reaction
(1) The principal limitation of the technique is that the DNA sequences at the ends
of the region to be amplified must be known so that primer oligonucleotides can
be synthesized.
(2) In addition, sequences longer than about 5,000 base pairs cannot be replicated
efficiently by conventional PCR procedures, although there are modifications of
PCR that allow longer fragments to be amplified. On the other hand, many
applications require amplification of relatively small fragments.
6. Designing the Oligonucleotide Primers for a PCR
The primers are the key to the success or failure of a PCR experiment. If the primers
are designed correctly the experiment results in amplification of a single DNA
fragment, corresponding to the target region of the template molecule.
If the primers are incorrectly designed the
experiment will fail, possibly because no
amplification occurs, or possibly because the
wrong fragment, or more than one fragment,
is amplified
Each primer must, of course, be complementary (not identical) to its template strand
in order for hybridization to occur, and the 3’ ends of the hybridized primers should
point toward one another.
The DNA fragment to be amplified should not be greater than about 3 kb in length
and ideally less than 1 kb. Fragments up to 10 kb can be amplified by standard PCR
techniques, but the longer the fragment the less efficient the amplification and the
more difficult it is to obtain consistent results.
Length of the primers
If the primers are too short they might hybridize to non-target sites and give
undesired amplification products.
Why not simply make the primers as long as possible?
The length of the primer influences the rate at which it hybridizes to the template
DNA, long primers hybridizing at a slower rate.
In practice, primers longer than 30-mer are rarely used.
Working Out the Correct Temperatures to Use
The annealing temperature is the important one because, again, this can affect the
specificity of the reaction.
The ideal annealing temperature must be low enough to
enable hybridization between primer and template, but high
enough to prevent mismatched hybrids from forming. This
temperature can be estimated by determining the melting
temperature or Tm of the primer–template hybrid.
Tm = (4 x [G + C]) + (2 x [A + T])°C
in which [G + C] is the number of G and C
nucleotides in the primer sequence, and
[A + T] is the number of A and T nucleotides.
After the PCR: Studying PCR Products
Three techniques are particularly important:
1.
2.
3.
Gel electrophoresis of PCR products
Cloning of PCR products
Sequencing of PCR products.
Gel Electrophoresis of PCR Products
A band representing the amplified
DNA may be visible after staining,
or if the DNA yield is low the
product can be detected by
Southern hybridization. If the
expected band is absent, or if
additional
bands
are
present,
something has gone wrong and the
experiment must be repeated.
the presence of restriction sites in the amplified region of the
template DNA can be determined by treating the PCR product with
a restriction endonuclease before running the sample in the
agarose gel (Figure 6.7). This is a type of restriction fragment
length polymorphism (RFLP) analysis and is important both in
the construction of genome maps and in studying genetic
diseases.
Cloning PCR Products
1. A special cloning vector which carries
thymidine (T) overhangs and which can
therefore be ligated to a PCR product
Special vectors of this type have also been
designed for use with the topoisomerase
ligation method, and this is currently the
most popular way of cloning PCR products.
(2) A second solution is to design primers that contain
restriction sites. After PCR the products are treated
with the restriction endonuclease, which cuts each
molecule within the primer sequence, leaving stickyended fragments that can be ligated efficiently into a
standard cloning vector
Conventional PCR
vs. Real-Time PCR
Conventional PCR
(1)Most conventional PCR-based tests used in molecular biology
laboratories needed to be performed in dedicated spaces to
control or reduce contamination that was always a threat for
producing false positive test results.
(2)Conventional PCR assays also require multiple manipulations
including (i) initial amplification of target nucleic acid, (ii)
detection of amplified product by gel electrophoresis, and (iii)
then confirmation by an alternative method such as southern
blotting or chemiluminescence techniques.
(3)In general, a conventional PCR assay would require a minimum of
at least 4-6 hours from the time that extracted nucleic acid is
placed into a thermal cycler to begin amplification to subsequent
product detection.
Real-Time PCR
1. Real Time (amplification and detection simultaneously): The new
instruments combine thermo cycling or target DNA amplification with
the ability to detect amplified target by fluorescently labeled probes
as the hybrids are formed (i.e., detection of amplicon in real time).
2. Avoidance of Cross Contamination: As both amplification and product
detection can be accomplished in one reaction vessel without ever
opening the major concern of cross contamination of samples with
amplified product associated with conventional PCR assays is greatly
lessened
3. Quantitate PCR Products: These instruments are not only able to
measure amplified product (amplicon) as it is made, but because of
this capability, they are also able to quantitate the amount of product
and thereby determine the number of copies of target in the original
specimen.
4. Reduce Time: The amount of time to complete a real-time PCR-based
assays because the time required for the post-PCR detection of
amplified product is eliminated by the use of fluorescent probes. Also,
some systems are able to perform rapid thermal cycling based on
instrument design, detecting product in as little as 20 to 30 minutes.
Real-Time PCR Enables the Amount of Starting
Material to be Quantified
• The amount of product that is synthesized during a set
number of cycles of a PCR depends on the number of DNA
molecules that are present in the starting mixture.
• If there are only a few DNA molecules at the beginning of
the PCR then relatively little product will be made, but if there
are many starting molecules then the product yield will be
higher.
• This relationship enables PCR to be used to quantify the
number of DNA molecules present in an extract.
Carrying Out a Quantitative PCR Experiment
In quantitative PCR (qPCR) the amount of product synthesized during a test PCR is
compared with the amounts synthesized during PCRs with known quantities of
starting DNA.
Although easy to perform, this type of qPCR is imprecise, because
large differences in the amount of starting DNA give relatively
small differences in the band intensities of the resulting PCR
products.
Real-time PCR
1. A dye that gives a fluorescent signal when it binds to
double-stranded DNA can be included in the PCR mixture.
This method measures the total amount of double-stranded
DNA in the PCR at any particular time, which may overestimate the actual amount of the product because
sometimes the primers anneal to one another in various
non-specific ways, increasing the amount of double-stranded
DNA that is present.
2. A short oligonucleotide called a reporter
probe, which gives a fluorescent signal when it
hybridizes to the PCR product, can be used.
Because the probe only hybridizes to the PCR
product, this method is less prone to
inaccuracies caused by primer-primer
annealing.
Each probe molecule has pair of labels.
A fluorescent dye is attached to one end of the oligonucleotide, and a quenching
compound, which inhibits the fluorescent signal, is attached to the other end
Real-Time PCR can also Quantify RNA
Real-time PCR is often used to quantify the amount of DNA in an extract, for
example to follow the progression of a viral infection by measuring the amount
of pathogen DNA that is present in a tissue
PCR Amplification of Full-Length cDNAs
Gene Synthesis by PCR