Download PCR

Molecular Biology
(MLMB-201)
Department of Medical Laboratory Technology
Faculty of Allied Medical Science
Lecturer:
Dr. Mohamed Salah El-Din
Intended Learning Outcomes (ILO’s):
Molecular biology course provides an overview of the molecular basis to cell structure and function.
This course focuses on the structure, biosynthesis and function of DNA and RNA on the molecular level and
how these interact among themselves and with proteins. Molecular biology techniques are essential for
modern biological and medical research. This course will give you an introduction to DNA and RNA standard
techniques.
Student will have basic knowledge of:
• Cell organization.
• DNA structure and function.
• DNA Extraction.
• RNA structure and function.
• RNA Extraction.
• Gene expression and protein biosynthesis.
• Agarose gel electrophoresis for DNA/RNA; and SDS-PAGE for protein.
• Polymerase Chain Reaction (PCR) – Theory, Types, Application.
• Gene library and screening
• DNA sequencing
The Polymerase Chain Reaction
(PCR)
PCR: is DNA replication in a test tube
What is the Polymerase Chain Reaction?
• It’s a means of selectively amplifying a particular segment of DNA.
• The segment may represent a small part of a large and complex
mixture of DNAs: e.g. a specific exon of a human gene.
• It can be thought of as a molecular photocopier.
How Powerful is PCR?
• PCR can amplify a usable amount of DNA (visible by gel electrophoresis) in ~2 hrs.
• The template DNA need not be highly purified — a boiled bacterial colony.
• The PCR product can be digested with restriction enzymes, sequenced or cloned.
• PCR can amplify a single DNA molecule, e.g. from a single sperm.
The Basics of PCR Cycling
• 30 – 35 cycles each comprising:
– denaturation (95°C), 30 sec.
– annealing (55–60°C), 30 sec.
– extension (72°C), time depends
on product size.
What’s in the Reaction?
• Template DNA
• Reaction buffer: (Tris, ammonium ions (and/or potassium ions),
magnesium ions, bovine serum albumin)
• Nucleotides (dNTPs)
• Primers
• DNA polymerase (usually Taq)
Primers
•
Must have some information about sequence flanking your target
•
Primers provide specificity
•
Complementary to opposite strands with 3’ ends pointing towards each other
•
Should have similar melting temperatures
•
Be in vast excess
Melting temperature
 TmoC = 2(A/T) + 4(G/C)

TmoC Temperature at which half possible H bonds are formed
Designing PCR Primers
• Primers should be ~20 bases long.
• The G/C content should be 45–55%.
• The annealing temperatures should be within 1°C of one another.
• The 3´-most base should be a G or C.
• The primers must not base pair with each other or with themselves
or form hairpins.
• Primers must avoid repetitive DNA regions.
Primers That Form Hairpins
Primers That Form Dimers
• A primer may be self-complementary
and be able to fold into a hairpin:
5´-GTTGACTTGATA
|||| | T
3´-GAACTCT
• A primer may form a dimer with itself or
with the other primer.
5´-ACCGGTAGCCACGAATTCGT-3´
|| ||| || || |
3´-TGCTTAAGCACCGATGGCCA-5´
• The 3´ end of the primer is base-paired,
preventing it annealing to the target
DNA.
• Primer dimers can be an excellent, but
unwanted, substrate for the Taq polymerase.
Thermus aquaticus DNA polymerase (Taq):
•
Not permanently destroyed at 94ºC
•
Optimal temperature is 72ºC
Problems with Taq
•
Does not have proof readng ability
•
Error rate 1 in 2 X 104 bases
•
Seems rare but can be recovered in cloning
a single molecule
•
Newer polymerases have high fidelity
Thermus aquaticus
(Taq)
Templates for PCR
PCR Templates could be:
 Small amount of template (DNA/cDNA)


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
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 In theory a single molecule
 Do not need to isolate sequence of interest
 DNA template need not be highly purified
 DNA is stable in absence of nucleases
Optimizing the PCR Reaction

Annealing temperature of the primers.

The concentration of Mg2+ in the reaction.

The extension time.

The denaturing and annealing times.

The extension temperature.

The amount of template and polymerase - “more is less”.
Dried blood
Semen stains
Vaginal swabs
Single hair
Fingernail scrapings
Insects in Amber
Egyptian mummies
Buccal Swab
Toothbrushes
Fidelity of the Reaction
• Taq DNA polymerase lacks the 3´→ 5´proof-reading activity commonly
present in other polymerases.
• Taq mis-incorporates 1 base in 104.
•
A 400 bp target will contain an error in 33% of molecules after 20 cycles.
•
Error distribution will be random.
Do Errors Matter?
• Yes, if you want to clone the amplified DNA - an individual molecule may
harbour several mutations.
• No, if you want to sequence the amplified DNA or cut it with restriction enzymes.
• Use a proof-reading thermo-stable enzyme rather than Taq.
How Big A Target?
• Amplification products are typically in the size range 100-1500 bp.
• Longer targets are amplifiable — >25 kb. :
• Requires modified reaction buffer, cocktails of polymerases, and longer
extension times.
• Limited by the integrity of the starting target DNA — > 50 kb.
Can PCR Amplify RNA?
• Not directly - the DNA polymerase requires a DNA template and will not
copy RNA.
• mRNA can first be copied into cDNA using reverse transcriptase.
• cDNA is a template for PCR.
RT-PCR
It is a reverse-transcriptase PCR test
to detect RNA and is composed of the
same 3 basic parts as PCR and an
additional
step
using
reversetranscriptase enzyme to synthesize
comple-mentary DNA from the target
RNA. The complementary DNA is then
run in the PCR test.
http://www.bio.davidson.edu/courses/Immunology/Flash/RT_PCR.html

Avian myeloblastosis virus (AMV) or Moloney murine
leukemia virus (M-MLV or MuLV) reverse transcriptases
are generally used to produce a DNA copy of the RNA
template using either random primers, an oligo(dT)
primer or a sequence-specific primer. Alternatively, some
thermostable DNA polymerases (e.g., Tth DNA
polymerase) possess a reverse transcriptase activity,
which can be activated under certain conditions, namely
using manganese instead of magnesium as a cofactor.
After this initial reverse transcription step has produced
the cDNA template, basic PCR is carried out to amplify
the target sequence.

The quality and purity of the starting RNA template is
crucial to the success of RT-PCR. Either total RNA or
poly(A)+ RNA can be used as the starting template, but
both must be intact and free of contaminating genomic
DNA. Specific capture of poly(A)+ RNA will enrich a
targeted message so that less of the reverse
transcription reaction is needed for the subsequent
amplification. The efficiency of the first-strand synthesis
reaction, which can be related to the quality of the RNA
template, will also significantly impact the results of the
subsequent amplification.
Template Considerations
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Procedures for creating and maintaining an RNase-free
environment are mandatory . The use of an RNase
inhibitor (e.g., Recombinant RNasin® Ribonuclease
Inhibitor) is strongly recommended.
For optimal results, the RNA template, whether a total
RNA preparation, an mRNA population or a synthesized
RNA transcript, should be DNA-free.
Using total RNA template levels in the range of 10pg–
1μg per reaction or poly(A)+ RNA template levels in the
range of 1pg–100ng.
Reverse Transcription Primer
Design

Selection of an appropriate primer for reverse
transcription depends on target mRNA size and the
presence of secondary structure. For example, a primer
that anneals specifically to the 3′-end of the transcript (a
sequence-specific primer or oligo(dT) primer) may be
problematic when reverse transcribing the 5′-ends of
long mRNAs or molecules that have significant
secondary structure, which can cause the reverse
transcriptase to stall during cDNA synthesis. Random
hexamers prime reverse transcription at multiple points
along the transcript. For this reason, they are useful for
either long mRNAs or transcripts with significant
secondary structure.

Whenever possible, it is recommended to use a primer that anneals
only to defined sequences in particular RNAs (sequence-specific
primers) rather than to the entire RNA population in the sample (e.g.,
random hexamers or oligo(dT) primer). To differentiate between
amplification of cDNA and amplification of contaminating genomic
DNA, design primers to anneal to sequences in exons on opposite
sides of an intron, so any amplification product derived from
genomic DNA will be much larger than the product amplified from
the target cDNA. This size difference not only makes it possible to
differentiate the two products by gel electrophoresis but also favors
the synthesis of the smaller cDNA-derived product (PCR favors the
amplification of smaller fragments).

Regardless of primer choice, the final concentration of the primer in
the reaction is usually within the range of 0.1–1.0μM, but this may
need to be optimized. We recommend using a final concentration of
1μM primer (50pmol in a 50μl reaction) as a starting point for
optimization.
Cycle Parameters

Efficient
first-strand
cDNA synthesis
can
be
accomplished in a 20 to 60 minute incubation at 37°C –
45°C using AMV reverse transcriptase. It is
recommended using a sequence-specific primer and
performing the reverse transcription reaction at 45°C for
45 minutes as a starting point. The higher temperature
will minimize the effects of RNA secondary structure and
encourage full-length cDNA synthesis. First-strand cDNA
synthesis with random hexamers and oligo(dT) primer
should be conducted at room temperature (20 – 25°C)
and 37°C, respectively.

An RNA denaturation step prior to initiation of the reverse
transcription reaction is desired. However, a denaturation step may
be incorporated by incubating a separate tube containing the
primers and RNA template at 94°C for 2 minutes. Do not incubate
AMV reverse transcriptase at 94°C; it will be inactivated. The
template/primer mixture can then be cooled to 45°C and added to
the RT-PCR reaction mix for the standard reverse transcription
incubation at 45°C.

Following the reverse transcription, it is recommended to do a 2minute incubation at 94°C to denature the RNA/cDNA hybrid,
inactivate AMV reverse transcriptase and dissociate AMV RT from
the cDNA.

Most RNA samples can be detected using 30 – 40 cycles of
amplification. If the target RNA is rare or if only a small amount of
starting material is available, it may be necessary to increase the
number of cycles to 45 or 50 or dilute the products of the first
reaction and reamplify.
Applications of PCR
• Mutation testing, e.g. cystic fibrosis.
• Diagnosis or screening of acquired diseases, e.g. AIDS.
• Detect tuberculosis without culturing.
• Genetic profiling in forensic, legal and biodiversity applications.
• Paternity testing.
• Site-directed mutagenesis of genes.
• Quantitation of mRNA in cells or tissues.
Variations on the basic PCR
Nested PCR – Nested PCR increases the specificity of DNA amplification, by
reducing background due to non-specific amplification of DNA. Two sets of
primers are being used in two successive PCR reactions. In the first reaction,
one pair of primers is used to generate DNA products, which besides the
intended target, may still consist of non-specifically amplified DNA fragments.
The product(s) (sometimes after gel purification after electrophoresis of the
PCR product) are then used in a second PCR reaction with a set of primers
whose binding sites are completely or partially different from the primer pair
used in the first reaction, but are completely within the DNA target fragment.
Nested PCR is often more successful in specifically amplifying long DNA
fragments than conventional PCR, but it requires more detailed knowledge of
the target sequences.
Multiplex-PCR - The use of multiple, unique primer sets within a single PCR
reaction to produce amplicons of varying sizes specific to different DNA
sequences. By targeting multiple genes at once, additional information may be
gained from a single test run that otherwise would require several times the
reagents and more time to perform. 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.
Allele-specific PCR - AS-PCR is used to determine the genotype of single
nucleotide polymorphisms (SNPs) (single base differences in DNA) by using
primers whose ends overlap the SNP and differ by that single base. PCR
amplification is less efficient in the presence of a mismatch, so the differences
in amplification resulting from different primers can be used to quickly
determine which primer matches the sample genotype.
RT-PCR – RT-PCR (Reverse Transcription PCR) is a method used to amplify,
isolate or identify a known sequence from a cellular or tissue RNA. The PCR
reaction is preceded by a reaction using reverse transcriptase to convert RNA to
cDNA. 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 and, if the genomic DNA sequence of a gene is known, 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 a RT-PCR
method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.
Quantitative PCR – Q-PCR (Quantitative PCR) is used to measure the quantity
of a PCR product (preferably real-time). It is the method of choice to
quantitatively measure 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. The method with currently the
highest level of accuracy is Quantitative real-time PCR. It is often confusingly
known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more
appropriate contractions. RT-PCR commonly refers to reverse transcription PCR
(see below), which is often used in conjunction with Q-PCR. QRT-PCR methods
use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA
probes, such as TaqMan, to measure the amount of amplified product in real
time.
Touchdown PCR – Touchdown PCR is 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 above the Tm of the primers used, while at the later cycles, it is a few
degrees 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.
Colony PCR - Bacterial clones (E. coli) can be rapidly screened for correct DNA
vector constructs. Selected bacterial colonies are picked with a sterile toothpick
from an agarose plate and dabbed into the master mix or sterile water. Primers (and
the master mix) are added, and the PCR is started with an extended time at 95˚C
when standard polymerase is used or with a shortened denaturation step at 100˚C
and special chimeric DNA polymerase.
RAPD PCR or AP-PCR: Random Amplification of Polymorphic DNA or
arbitrary primed PCR or DOP-PCR
RAPD reactions are PCR reactions, but they amplify segments of DNA which
are essentially unknown to the scientist (random).
In RAPD analysis, the target sequence(s) (to be amplified) is unknown. The
scientist will design a primer with an arbitrary sequence. In other words, the
scientist simply makes up a 10 base pair sequence (or may have a computer
randomly generate a 10 bp sequence), then synthesizes the primer. The
scientist then carries out a PCR reaction and runs an agarose gel to see if any
DNA segments were amplified in the presence of the arbitrary primer.
Asymmetric PCR: It is used to preferentially amplify one strand of the original DNA
more than the other. It finds use in some types of Sequencing and hybridization where
having only one of the two complementary stands is ideal. PCR is carried out as
usual, but with a great excess of the primers for the chosen strand. Due to 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.
Inverse PCR: It is a method used to allow PCR when only one internal sequence
is known (i.e. for amplification of regions flanking a known sequence). DNA is
digested, the desired fragment is circularise by ligation, then PCR using primer
complementary to the known sequence extending outwards.
Assignment:
As a part of the semester activity, a
group of students is selected every
week to prepare a short seminar about
his/her point of interest in one of the
lecture topics. That to be discussed
and evaluated during the next lecture.