4. Primer Design & Optiomization

Question 1

What are the characteristics of a good primer (for specific PCR)

Answer 1

1. should be unique
2. at least 15 bases long
3. no hairpin formations
4. melting temperature in the range of 52-62c
5. base compositon avoids long stretches of AG/CT

Question 2

why should primers be unique

Answer 2

primers should be highly specific and flank only the target
sequence to avoid mispriming

Question 3

mispriming is

Answer 3

When primers successfully anneal to other areas of the genome, the result is a band not of the correct (expected) size.

Question 4

why should primers be at least 15 bases long to be a good primer

Answer 4

Primer length has effects on uniqueness and melting/annealing temperature.

Generally, the longer the primer, greater chance that it is unique

the longer the primer, the higher melting/annealing temperature.

primer should be at least 15 bases to ensure uniqueness

Question 5

generally, the longer the primer, the greater chance that it is

Answer 5

unique and the higher the melting/annealing temperatures

Question 6

why should a good primer not have hairpin formations

Answer 6

if primers can anneal to themselves, or anneal to each
other rather than anneal to the template, PCR efficiency
can be decreased dramatically

Question 7

hairpin formations are when primers

Answer 7

dimerize or form secondary structures

Question 8

A primer melting temperature (Tm) should be in the range of

Answer 8

52C to 65 C

Question 9

Primer Melting Temperature, Tm is

Answer 9

the temperature at which half the primers are hybridized

Question 10

Annealing Temperature, T anneal is

Answer 10

the temperature at which primers anneal to the template DNA.

It can be calculated from Tm

Question 11

Both primers in a PCR reaction should have similar Tm (primer melting temperature) to

Answer 11

ensure similar hybridization kinetics during the annealing phase.

The maximum difference between the 2 primers T anneal
allowed is 3 °C. The closer they are, the better.

Question 12

The maximum difference between the 2 primers T anneal
allowed is

Answer 12

3 °C. The closer they are, the better.

Question 13

for PCR success base composition must avoid long stretches of AG/CT because

Answer 13

Base composition affects hybridization specificity and melting/annealing temperature.

Avoid long stretches of (A,G) purines or (C,T)pyrimidines: avoid
more than 3 C’s or G’s as these would promote mispriming in GC-rich templates

Question 14

Special Primers

Answer 14

1. primers with 5’ tags
2. degenerate primers
3. computer aided primer design

Question 15

things to do to get PCR to work when your PCR dosent work

Answer 15

1. check the DNA template
2. vary the MgCL
3. modify the annealing temperature

Question 16

if you PCR dosent work check the DNA template to see if

Answer 16

if it may be contaminated with proteins, phenolics, carbs etc which inhibit the enzyme

DNA may be degraded

DNA is in too low a concentration for effective amplification

Question 17

if you PCR dosent work you can vary the MgCL because

Answer 17

Low [MgCl2] increases specificity

high [MgCl2] stabilizes primer annealing and can increase
sensitivity, but can also decrease primer specificity

Question 18

Low [MgCl2] increases

Answer 18

specificity

Question 19

high [MgCl2] stabilizes

Answer 19

primer annealing and can increase sensitivity, but can also decrease primer specificity

Question 20

if you PCR dosent work you can modify the annealing temperature because

Answer 20

Temperature increases specificity of primer annealing by destabilizing base pair mismatches

temperature increases the sensitivity (and yield) of the reaction by stabilizing correct base pairing

Question 21

PCR Additives are

Answer 21

Reagents that are included in the PCR mix to improve the PCR product yield, specificity and reproducibility

Question 22

The positive effects of PCR additives are

Answer 22

varied from rxn to rxn and therefore, they must be empirically
tested for each combination of template and primers

Question 23

List of PCR additives

Answer 23

1.Dimethyl sulfoxide (DMSO)
2. Betaine (N,N,N-trimethylglycine)
3. Formamide
4. Non-ionic detergents
5. TMAC (tetramethylammonium chloride)
6. BSA (bovine serum albumin)
7. Glycerol
8. Polyethylene glycol (PEG)
9. 7-deaza-2ʹ′-deoxyguanosine
10. Gelatin

Question 24

Dimethyl sulfoxide (DMSO)

Answer 24

2-10% v/v may be necessary for amplification of some templates, however 10% DMSO can reduce Taq polymerase activity by up to 50% so it should not be used routinely.

DMSO is thought to reduce secondary structure and is particularly useful for GC rich templates.
DMSO often helps in amplifying products of >1kb

Question 25

DMSO often helps in amplifying products of

Answer 25

>1kb

Question 26

Betaine (N,N,N-trimethylglycine)

Answer 26

is generally used at a final concentration of 1.0-1.7M. It is used as an enhancer for GC rich template reactions

Question 27

Non-ionic detergents

Answer 27

e.g. Triton X-100, Tween 20 or Nonidet P-40 (NP-40) Stabilise Taq polymerase and may also suppress the formation of secondary structure. Neutralizes the effect of SDS which is used in DNA extraction but inhibits PCR by reducing polymerase activity

Question 28

TMAC (tetramethylammonium chloride)

Answer 28

is generally used at a final concentration of 15-100mM to eliminate non-specific priming

Question 28

Polyethylene glycol (PEG)

Answer 28

Used when DNA template concentration is very low It promotes macromolecular association by solvent exclusion ie. the Taq can ‘find’ the DNA

Question 29

Glycerol

Answer 29

improves the amplification of high (G+C) templates

Question 30

Gelatin

Answer 30

stabilize Taq DNA polymerase during PCR, which generally increases the yield

Question 31

Multiplex PCR

Answer 31

Multiple primer pairs are added in the same tube to do the PCR Good for amplifying multiple sites Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction Amplicon sizes should be different enough to form distinct bands when visualized by gel electrophoresis. Design difficulty Melting temperatures should be similar No dimer formulation Used for: Diagnostics

Question 32

Multiplex PCR is used for

Answer 32

diagnostics

Question 33

Nested PCR

Answer 33

2 sets of amplification primers USES: Diagnostics, allele identification

Question 34

Quantitative PCR (Q-PCR)

Answer 34

measure the quantity of a PCR product (commonly in real-time) Uses: Expression studies, diagnostics to detect viral load or microbial risk assassment

Question 35

Quantitative PCR (Q-PCR): uses

Answer 35

Expression studies, diagnostics to detect viral load or microbial risk assassment