Primers in biology

Primers

Kinds of primers

Parameters for primer designing

Primer

Short string of nucleotides which attaches to DNA templet

Serves as starting point for DNA synthesis

In the cell, Primase produces primer complementary to the DNA

segment being copied

PCR primer- short, single stranded, synthetically synthesized

oligonucleotides

Usually 18-25 nucleotides

Types of Primer
1. Single primer/ Probes
Used to amplify and label DNA fragment for sequencing reactions
Probes for southern hybridization
2. Primer pair (F/R)
Primer pair limits the range of DNA amplified during reaction
Error rate one in 100 base pairs

3. Degenerate primers

Mixtures of similar but not identical primers

Convenient

Often

for amplification of same gene in different organisms

used when primer designing is based on amino acid sequence

Reduced

specificity

Commonly

Designed

Used for microbial ecology studies

by aligning gene sequences in GenBank

PCR Reaction
Initial Denaturation
Denaturation
Annealing
Extension
Final Extension

Primer designing tools

Primer 3

Gene fisher interactive designing

PCR now

Bi Search

NCBI- Primer BLAST

Beacon designer

Primer Premier

Primer Plex

NCBI Primer BLAST

Uses primer 3 for designing

Submits primers to BLAST search against User Selected database

BLAST search automatically analyses primer pair that can cause

amplification of targets other than input templet

Primer designing parametrs

Length range
% GC Range
Tm Range
3 end stability
5 end stability

DG primer
DG Hairpin

Critical considerations for primer

design
1.

Primer length

Optimal length 18-22 bp

2. Primer Melting temperature (Tm)

-Temperature at which one half of the DNA duplex will dissociate to become single stranded
-Tm range 52-580 c ideal (DNA pol optimal temp)
-Tm above 65 degree responsible for secondary annealing
-Tm depends on GC content
Tm= 2(A+T) + 4 (G+C)
-Tm > annealing temperature =mishybridization/ misannealing
-Tm < annealing temp =failed annealing

3. Primer Annealing temperature(Ta)

Ta= 0.3 X Tm (Primer) + 0.7 X Tm ( Product)
-Higher
-Lower

Ta = insufficient primer template hybridization

Ta= non specific products

4. GC Content
-Ideal should be 40-60%
5.GC Clamp at 3 end
- Presence of G/C bases within last five nucleotides at 3 end
-More

than 3 G/C nucleotides should be avoided

6. Primer Secondary Structures

- produced by intramolecular or intermolecular interaction leading to poor or no
yield
-Reduce

availability of primers for reaction

6.1 Primer Dimer

-Annnealing

of primers with other primers in mixture (Same or reverse direction

6.2 Hairpins
- Formed by intramolecular interactions within the primer

Primer Dimer

6.3 Self Dimer

-Imtermolecular

interactions between two primers where primer is homologous

to itself

6.4 Cross Dimer

- Intermolecular interaction between sense and antisense primer

7. Repeats
-Dinucleotide

occurring many times consecutively (ATATATAT)

-Mononucleotide
-Leads

repeats should be avoided

to loop formation/ mishybridization

8. Runs
-Long

runs of single base should be avoided

-ATGCCCCCCATACC
-Maximum

accepted run 4 bp

9. 3 end stability
-Unstable

3 end results in false priming

10. Templet Secondary Structure

-Single
-Can

strand is highly unstable

fold into secondary structures (Conformations)

-Designing

primers in region of templet that do not form stable secondary

structures
11. Cross Homology
-Use

BLAST to test specificity

-Perform

BLAST on Templet for identification of regions with significant cross

homology
-Avoid

regions of homology

Applications of PCR Primers

Amplification

Sequencing

Probes