Primers Designing in PCR

1. PRESENTED BY
Sourin De
M PHARM 1st YEAR
PHARMACOLOGY
PRIMER DESIGNING IN PCR

2. PCR- INTRODUCTION

3. • An in-vitro technique that takes specific sequence
of DNA of small amount and amplifies it to be used
for further testing.
• Able to a identify and manipulate DNA,
• Detect infectious organisms, including the viruses
that cause AIDS, hepatitis, tuberculosis
• Detect genetic variations, including mutations in
human genes
• Purpose : To amplify a lot of double-stranded DNA
molecules (fragments) with same (identical) size
and sequence by enzymatic method and cycling
condition

4. STEPS
I. Denaturation

5. II. Annealing
• Primers bind to their complementary sequences
• The primers dictate the part of the template that will
be amplified
• Temp: 50-70C (dependent on the melting
temperature of the expected duplex)

6. III. Primer extension
• DNA synthesis occurs (68–72C)
• 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

7. Cycling
• 3 Steps: One cycle one copy of double-stranded
DNA has been replicated into two copies.
• DNA 1 copy
• After PCR

8. PRIMERS
 Short ssDNA sequence (15-20 nucleotide in
length).
 Designing a good pair of oligonucleotide primers
is imp.
 2 types of primer
 Forward primer→anneal to anti sense strand of
dsDNA (3’-5’)
 Reverse primer→ anneal to sense strand of
dsDNA(5’-3’)

9. Forward and reverse primer

10. PCR PRIMER DESIGN
CONSIDERATION
 Primer specificity

11. • Primer length
Should be in a range of 18-24 bp is optimal for
PCR technq

12. • Annealing and melting temperature
 Annealing temp→ temp at which primer binds to
template DNA
 should be lower than the Tm by 5c to 10c
 Melting temp→ temp at which 50% of dsDNA is
denatured into ssDNA

13. •Primers with a Tm of 52-58c is preferred.
 Temperature should not exceed 60c
 GC content of the sequence gives indication of primer
Tm.
 Formula for Tm calculation
Tm=[4(G+C)+2(A+T)]C

14. • GC content
 Indicates the number of G and C in the primer as a
percentage of total bases. It should be 60%.
 GC clamp
 Presence of guanine or cytosine base in the last 5
bases (3’) end of primer
 improve specificity of
primer binding to complementary
sequence.
 Not recommended to include >2G
or C bases in last 5 bases ,because it
can increase Tm and affect primer
specificity

15. Primer secondary structure
 Hairpins: formed by intramol interaction within the primer.
 Primer dimer: formed by intermol interaction between primer.
Runs and repeats
 Repeats→ dinucleotide occuring many times consecutively and it
should be avoided.
Eg: ATATATAT( max no is 4)
 Runs→ long runs of single base should be avoided
Eg: AGCGGGGGTGGGG ( runs = 5 &4 ) → maximum is 4

16. .

17. .
 Materials: algorithm used to design a set of 600
primers surrounding CDKN2A locus
 Method: PAMP design to identify structural variation
in genome.
 Melting temperature: 57c
 Discussion: detect deletions within a given region.
 Advantage: deletion,insertion, translocation…can be
assayed by using PAMP.

18. ,
THANK YOU