PRIMER DESIGNING FOR PCR

WHAT IS PCR?
Polymerase chain reaction is an invitro technique which is use to amplify DNA sequences. The
method involves using short DNA sequences called primers to select the portion of the genome
to be amplified.

The temperature of sample is repeatedly raised and lowered at different points to help replication
enzyme copy the target DNA sequence.

The technique can produce billion of copies of target sequence in few hours.

HISTORY OF PCR
Kary Mullis invented PCR technique in 1985 while working as a chemist at the Cetus
Corporation, a biotechnology firm in Emeryville, California.

In 1993 Mullis was awarded noble price for his work on PCR.

REQUIREMENTS FOR PCR

 Taq polymerase
 DNA template
 Primers
 Deoxynucleosides
 Triphosphates
 Buffer solutions
 Divalent cations

USES OF PCR
 Genotyping
 Cloning
 Mutation detection
 Sequencing
 Microarrays
 Forensics
 Paternity testing
 Used to identify and explore relationships among species in the field of evolutionary
biology
  In anthropology, used to understand ancient human migration pattern
 In archaeology, used to spot ancient human race
 DNA amplifications
 Environmental and food safety testing
 Site-directed mutagenesis

ADVANTAGES OF PCR

 Quickly performed in 4-8 hours

 It is highly sensitive
 It is a versatile technique that can be adapted to wide range of applications
 PCR allows for the precise quantification of DNA or RNA in the sample
 Ability to test for anti-microbial resistance
 It is relatively cost effective technique compared to traditional methods of DNA analysis
 PCR is widely accessible to researchers, clinicians and in laboratories

DISADVANTAGES OF PCR
 It is sensitive to inhibitors present in the sample
 It is used to amplify DNA fragments up to few kilobases in length
 It is highly susceptible to contamination which may lead to false positive result
 Become less cost effective when performed with multi organism PCR

TYPES OF PCR
 Real-time PCR
 Reverse transcriptase PCR
 Multiplex PCR
 Nested PCR
 Long range PCR
 Single-cell PCR
 In –situ PCR
 Assemble PCR
 Mini primer PCR
 Solid phase PCR
 Touch down PCR
 Methylation specific PCR
  Hot start PCR
 Colony PCR
 Overlap extension PCR etc

PCR PROCESS STEPS

Polymerase Chain Reaction (PCR) is a technique used to amplify trace amount of DNA and
RNA from a sample.

A PCR thermal cycler is used to produce large amounts required for research

The PCR process can be used for a wide variety of laboratory and clinical applications and
purposes.

1. Forensic labs use it to analyse DNA sample from crime scene.

2. Clinical health care labs use it to diagnose patients infected from virus.
3. Pharmaceutical research labs use it to analyse duplicate DNA and RNA samples for use
in manufacturing of drugs and vaccines

SAMPLE COLLECTION AND PREPARATION

4 components are needed for PCR process:

 A DNA or RNA sample (from saliva, urine, blood etc)

 DNA primer ; short single stranded DNA that promote synthesize of complementary
strand of nucleotide
 DNA polymerase; an enzyme that aids in synthesize of complementary strand pf DNA
 Nucleotide solution mix containing Adenine(A), Thiamine(T), Cytosine(C), Guanine(G)
to build duplicate DNA strand.

After setup of PCR, amplification is done through thermal cycler and analyzation of product
is done using agarose gel electrophoresis and other methods to identify presence and size of
amplicons

Then data is analysed to draw conclusions about presence or absence of target sequence

THE STEPS EXPLAINED

The process begins with a segment of DNA sample placed in a suitable tube along with reagents and
chemicals mentioned above.

The tube is placed in PCR machine or thermal cycler.

This include 3 steps; denaturation, annealing and extension

 i. DENATURATION

The solution is added in the tube and heated to at least 90-98 C using a thermal cycler.

The heat breaks the hydrogen bonds of the sample strands and separates the DN into single
strand.

The duration of the process is about 1-2 minutes.

ii. ANNEALING

The sample mixture is then cooled to between 45 to 60 C and allows the primers and the DNA
polymerase to bind to individual strands of DNA that is separated by the heat.

At this point, the nucleotides will attach to the strands of the DNA. Or we can say that hydrogen
bond reforms

It is also known as renaturating.

iii. EXTENSION

The temperature is shifted to 70 C which is ideal for polymerase.

Elongation process continues here, where polymerase add Dntp from 5’to 3’ and read the
template from 3’to 5’.

This cycle is repeated from 35 to 40 times using thermal cycler which automatically repeats the
heating and cooling cycles of process.

iv. ANALYSIS WITH ELECTROPHEROSIS

Once the PCR process is completed, the resulting amplified segments generated can be compared
to other segments from the known source.

The PCR generated sequences are placed in a separating gel.

Electric current is run through the gel and the sequences within gel forms band that resembles a
ladder, according to their electrical charge and molecular size. It is known as gel electrophoresis.

PRIMER SPECIFICATIONS
Now we are going to discuss about specifications of the primer used in PCR

Good primer is essential for successful reactions.

Some specifications are described below with high yield.

 i. PRIMER LENGTH

The optimal length of PCR primer is 18-22 bp. This length is long enough for adequate
specificity and short enough for primer to bind easily to template at temperature

ii. PRIMER MELTING TEMPERATURE

It is the temperature at which one half of DNA duplex will dissociate to become single
stranded and indicates duplex stability.

Primers with melting temperatures in the range of 52-58 C generally produce best results.

Primers with the melting temperature above 65 C have a tendency for secondary annealing.

The GC content of the sequence gives a fair indication of the primer T m .

Formula for primer T m calculation:

Melting Temperature T m (K)={ΔH/ ΔS + R ln(C)},
Or Melting Temperature T m ( o C) = {ΔH/ ΔS + R ln(C)} - 273.15 where
 ΔH (kcal/mole):
H is the Enthalpy. Enthalpy is the amount of heat energy possessed by
substances. ΔH is the change in Enthalpy.
 ΔS (kcal/mole):
S is the amount of disorder a system exhibits is called entropy. ΔS is
change in Entropy.
iii. PRIMER ANNEALING TEMPERATURE

The primer melting temperature is the estimate of the DNA -DNA hybrid stability
and critical in determining the annealing temperature.
Too high T a will produce insufficient primer-template hybridization resulting in low
PCR product yield.
Too low T a may possibly lead to non-specific products caused by a high number of
base pair mismatches, Mismatch tolerance is found to have the strongest influence
on PCR specificity.
T a = 0.3 x T m (primer) + 0.7 T m (product) – 14.9
where,

T m (primer) = Melting Temperature of the primers

T m (product) = Melting temperature of the product
 iv. GC CONTENT
The GC content of the primer should be 40 -60%
v. GC CLAMP
The presence of G or C bases within the last five bases from the 3' end of primers
(GC clamp) helps promote specific binding at the 3' end due to the stronger bonding
of G and C bases.
More than 3 G's or C's should be avoided in the last 5 bases at the 3' end of the
primer.
vi. REPEATS
A repeat is a di-nucleotide occurring many times consecutively and should be
avoided because they can misprime.

For example:

ATATATAT. A maximum number of di -nucleotide repeats acceptable in an oligo is

4 di-nucleotides

vii. RUNS

Primers with the long runs of a single base should generally be avoided because
they can misprime.

For example;

AGCGGGGATGGG has runs of base ’G’ of value 4 and 5. A maximum number of

runs accepted are 4bp.

PRIMER DESIGNING TOOLS

To design a primer we use some different software and tools.

 Primer premier
 PrimerQuest
 OligoAnalyzer
 NCBI primer-BLAST
 Primer3
 PrimerPlex
 PRIMO
Internet site{s)
http://biobase.dk/index.html

http://bcf.drl,arizona.edu/gcg.html

http://www.biodisk.com/

http://www.oligo.net

http://alces.med.umn.edu/webprimers.html

http://www.willamstone.com/

http://doprimer.interactiva.de/

http://www .genome. wi. mit. edu/cgi-bin/primer/primer3_www.cgi

http://d t.imgen.bcm.tmc.edu 9331 se~util/se~util. html

http://www.med.jhu.edu/medcenter/primer/primer.cgi

PROCEDURE
Search NCBI on Google
Search gene of interest i.e. suppose that we have pqqc gene
Search it in the bar
Select the particular gene and get sequence of that gene
Go on FASTA format and get the sequence of that gene
Then we copy the FASTA sequence and go on BLAST website
We select nucleotide BLAST and in it we paste the FASTA sequence
Run the BLAST nucleotide
We get many downloaded files in BLAST format
Select gene sequences and download
Copy the download
Then we will go on CLUSTALW website
Here we will see a box
Place the sequence from BLAST in it
Then execute multiple sequence alignment
Then go to justBio and sign in there
Open oligocalc after signing in the justbio
Then paste the forward and reverse sequence and press calculate
It gives the results like GC content, length, temperature etc
Then we go to primer3 and paste the sequences there and run it
 It will design primers for PCR
Our primer is ready

This is how primer designing for PCR is done

You can also look the process on the given link below;

https://youtu.be/5QgBUZw1Hxc?si=DODUwvldsJv89Ix -