Moustafa Alkhalaf

01/02/2016
*1983 Kary Mullis of Cetus Corp invents
PCR (Nobel Prize in 1993).
*1985 First paper describing PCR using
Klenow fragment of DNA polymerase
(Saiki et al).
*1988 First paper describing the use of
Taq DNA polymerase in PCR.
- PCR is a technique that allows rapid
amplification of a specific target of nucleic acid.

- It requires only some of the components of the

complex replication machinery to copy short
fragments of nucleic acid in a simple buffer
system in a test tube.
- PCR consists of a series of repeated
temperature changes, called cycles (25 and
40).
- Each cycle (three temperature shifts):
1- Denaturation
2- Annealing
3- Extension
Typical thermal cycler program

Initial DNA denaturation at 94°C for 5 minutes :

Denaturation at 94°C for 1 minute

Annealing at 55°C for 1 minutes 30 cycles

Extension at 72°C for 1 minutes

Final extension at 72°C for 2 minutes

Amplicons
Theoretical accumulation of PCR products (20 cycles)
Kinetics of accumulation of the target product during PCR:

E: is the early phase of primer

scanning and initial product
formation.

M: is the middle phase during

which product accumulates in
an exponential manner.

L: is the late phase or plateau

where product accumulation
is suboptimal.
Target
Deoxynucleotides (dNTPs)
Forward primer & reverse primer
DNA polymerase
PCR buffer
Target:
- That contains the region of the nucleic acid
fragment to be amplified.
- Target sequence can be DNA or RNA.
Deoxynucleotide triphosphates (dNTPs):
- 200μM
Modified nucleotides:
- labelling of PCR products
- secondary structure resolution
- prevention of contamination
- DNA sequencing
- random mutagenesis
DNA polymerase:
- DNA-dependent DNA polymerases
- Thermostable DNA polymerases (Taq DNA polymerase)
- Optimum temperature 72-75oC
- Always reads 5’3’
- Can have 3’5’ exonuclease activity
- RNA-dependent DNA polymerases (reverse transcriptase) for RT-
PCR
Tris-HCl:
- dipolar ionic buffer
- pH varies during PCR
- higher fidelity at lower pH
MgCl2
- Taq is dependent upon the presence of Mg2+ and shows its highest
activity at 1.2-1.3mM Mg2+
- The free Mg2+ concentration is affected by the dNTP concentration
- The magnesium can also affect the fidelity of DNA polymerases.
KCl:
assist primer–template annealing.
Gelatin:
stabilises enzyme.
Denaturing agents:
alters Tm
Primers:
- 100nM
- Anneal to single-stranded DNA template
- Provide initiation site for extension of new DNA
- Forward primer anneals to DNA anti-sense strand
- Reverse primer anneals to DNA sense strand

DNA sense strand = 5' - 3'

DNA antisense strand = 3' - 5'
 PCR Primer Design
• 15 -30 bases
• Balance of bases
• No base repetition of more than 3-4
• GC Content 40-60%
• Melting temperature (Tm) between 55° and 65°C
- Melting temperature (Tm): the temperature at which half the primers
are annealed to the target region.

Tm = (Number of G+C) × 4°C + (Number of A+T) × 2°C

- Melting temperature of a primer is an indicator of the annealing

temperature (normally 5oC below Tm)
PCR Primer Design…
Complementarity:
-not self-complementary
-not complementary to each other
-avoid complementarity at 3’ end
-avoid runs of ≥ 3 G or C at 3’ end
-avoid T at 3’ end
possibility of secondary structures:
- Hairpins
- primer- dimer
Avoid Cross Homology

There are many computer programs help in designing primers:

NCBI Primer design tool http://www.ncbi.nlm.nih.gov/tools/primer-blast/
Primer3 http://frodo.wi.mit.edu/primer3/
- Used when the DNA sequence is not known but amino acid
sequence data are available from the protein encoded by the
target gene.
- A mix of oligonucleotide sequences in which
some positions contain a number of possible bases,
giving a population of primers with similar
sequences that cover all possible nucleotide
combinations for a given protein sequence.

-Degeneracy should be avoided at last 3 nucleotides

at 3’ end.

IUPAC Codes
- RNA can be amplified by including a simple reverse transcription
(RT) step prior to PCR.
- Reverse transcriptase (RNA-dependent DNA polymerase) is used to
make a complementary DNA copy (cDNA) from RNA.
- cDNA then can be amplified.
Parameters might need to be optimized to increase yield,
sensitivity or specificity:
- Magnesium concentration
- Primer annealing temperature
- PCR primer design & concentration
-Template quality & quantity
Contamination problems
Source of contamination:
―Original template DNAs
― Cloned DNA molecules carrying the target gene
― Previously PCR-amplified molecules
Contamination prevention:
- Control reactions (during extraction, during PCR)
- Pipettes and tips
- Uracil N-glycosylase
- UV irradiation
- Designated PCR set-up areas:
Minimum of three work areas should be used:
1. DNA-free
2. Sample preparation
3. Thermal cycling and detection
There are many ways to analyse PCR products depending
upon the information required:
― the presence or absence of the target DNA sequence.
― the length of the amplified fragment.
― the yield of PCR product to quantitate the amounts of
the starting DNA or RNA.
― Verification of initial amplification product:
• Direct DNA sequencing of PCR products
• Probe hybridization
• Restriction analysis of a PCR product
 Gel electrophoresis:
- Electrophoresis is a method of separating substances based on the
rate of movement while under the influence of an electric field.
- Molecules (such as DNA) can be made to move through a gel made
of agar or polyacrylamide. DNA fragments are then visualized in the
gel with a special dye.
- The presence or absence of PCR product and its size (length) can be
determined.
Touchdown PCR
Hot start PCR
Nested PCR
Multiplex PCR
Solid phase PCR
Touchdown PCR:
―A method for increasing specificity of PCR.
―The initial annealing temperature should be several degrees
above the estimated Tm of the primers.
― This ensure that only specific annealing takes place.
―The annealing temperature is then gradually decreased until it
reaches the calculated annealing temperature of the primers.
―Amplification is then continued using this annealing
temperature.

Example: reducing the annealing temperature by 1°C every 2

cycles moving from 65°C to 55°C over the first 20 cycles. The
reaction should then be completed by another 10 cycles at a 55°C
annealing temperature.
Hot Start PCR:
A technique that reduces
non-specific amplification
which may occur at low
temperature.
Different strategies:
1- Adding DNA polymerase after
the initial denotation step.
2- Polymerase activity can be
inhibited at room temperature
through different mechanisms.
3- Using wax beads
Nested PCR:
• To improve the sensitivity and the specificity.
• Two pairs of PCR primers are used.
• The first pair amplifies the locus as in any PCR experiment.
• The second pair of primers (nested primers) binds within
the first PCR product and produce a second PCR product
that will be shorter than the first one.
• Semi-nested PCR:
when only one
internal primer
Used in the second
Round.
Multiplex PCR:
- More than one target
sequence can be amplified
by including more than one
pair of primers in the
reaction.
- An optimal combination of annealing
temperature and buffer concentration is
essential in multiplex PCR to obtain
highly specific amplification products.
PCR is used when:
- there is insufficient nucleic acid within a sample for reliable
detection via direct hybridisation.
- other methods of etiological diagnosis are unsuitable.
- a rapid result is desired.
- a more defined identification is indicated.
Detecting of DNA and RNA
Quantifying DNA and RNA
Sequencing
Cloning and recombinant DNA techniques
Labeling DNA or RNA molecules
PCR based Mutagenesis
Gene expression studies
Diagnosis of infections
Epidemiological studies
Genotyping
Mutations detection (genetic testing, cancer
diagnosis)
Forensic applications