2. What is primer ?
◦ A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis.
◦ primers are short strands of RNA. A primer must be synthesized by an enzyme called primase,
which is a type of RNA polymerase, before DNA replication can occur.
◦ The primer therefore serves to prime and lay a foundation for DNA synthesis.
◦ The primers are removed before DNA replication is complete, and the gaps in the sequence are
filled in with DNA by DNA polymerases.
◦ In the laboratory, scientists can design and synthesize DNA primers with specific sequences that
bind to sequences in a single-stranded DNA molecule. These DNA primers are commonly used to
perform the polymerase chain reaction to copy pieces of DNA or for DNA sequencing.
◦
3. WHO DISCOVERED PRIMER?
◦ Arthur Kornberg a American Biochemist.
◦ In 1956
◦ Primer was discovered when he was
working on mechanism of DNA replication.
He discovered DNA polymerase enzyme and
a short sequence of nucleotides to initiate
replication.
4. Primer :
◦ Short synthetic oligonucleotide which is
used in many molecular technique PCR to
DNA sequencing.
◦ Used to synthesis DNA &lify the
specific gene of interest .
◦ In living organism, helps to initiate DNA
Replication process.
◦ 2 types of primer
1. Forward primer 5' to 3' direction
2. Reverse primer 3' to 5' direction
5. General rules for primer design
1.Primers;
◦ must be complementary to flanking sequences of target region
2.Primer Length:
◦ It is generally accepted that the optimal length of PCR primers is 18-22 bp.
◦ Too long – decrease the template binding efficiency.
◦ Too short - low specificity resulting in non-specific amplification.
6. 3. Primer Melting Temperature (Tm):
◦ The primer melting temperature (Tm) is the temperature at which half of the
primers separate from the template DNA
◦ Primers with Tm in the range of 52-58 o C generally produce the best results.
◦ Primers with melting temperatures above 65°C have a tendency
for secondary annealing,
◦ The GC content of the sequence gives a fair indication of the primer Tm.
◦ Tm = (G + C) * 4 + (A + T) * 2
◦ The difference of Tm should be < 5oC of the forward and reverse primers.
7. 4. Primer Annealing Temperature (Ta):
◦ The annealing temperature for primers is typically 45–68°C, and is based on
the melting temperature (Tm) of the primer pair
(Ta = Tm – 5)
◦ Too high Ta will produce insufficient primer-template hybridization resulting in
low PCR product yield.
◦ Too low Ta may possibly lead to non-specific products caused by a high
number of base pair mismatches.
8. 5. GC Content: The GC content (the number of G's and C's in the primer as a
percentage of the total bases) of primer should be 40-60%.
6. GC Clamp: The presence of G or C bases within the last five bases from
the 3' end of primers (GC clamp)
7.Runs: Primers with long runs of a should generally be avoided. For example,
AGCGGGGGATGGGG . maximum number of runs accepted is 4 bp.
9. 8. Repeats: A repeat is a di-nucleotide occurring many times continuously. For
example: ATATATAT. maximum acceptable number of di-nucleotide repeats is 4.
9. 3' End Stability: It is the maximum AG value of the five bases from the 3' end.
An unstable 3' end (less negative AG) will result in less false priming.
