8/13/2019 PCR 31-08-12

1/44

Morgan 1922 : 2000 genes in 4 chromosome of D.melanogaster

Avery, MacLeod & McCarty 1944 DNA is the geneticmaterial

Harsey Chase 1952 DNA is the genetic material

Delbruck, Chargaff, Crick, Monod DNA stracture

8/13/2019 PCR 31-08-12

2/44

Recombinant DNA technology / genetic EngineeringGene cloning

DNA sequencing

8/13/2019 PCR 31-08-12

3/44

PCR

Kary Mullis 1985

Coast of California drive

8/13/2019 PCR 31-08-12

4/44

PCR

8/13/2019 PCR 31-08-12

5/44

DNA in a Cell

8/13/2019 PCR 31-08-12

6/44

8/13/2019 PCR 31-08-12

7/44

8/13/2019 PCR 31-08-12

8/44

8/13/2019 PCR 31-08-12

9/44

AGAROSE GELELECTROPHORESIS

8/13/2019 PCR 31-08-12

10/44

8/13/2019 PCR 31-08-12

11/44

8/13/2019 PCR 31-08-12

12/44

PURIFYING NUCLEIC ACIDFRAGMENTS FROM GEL

8/13/2019 PCR 31-08-12

13/44

POLYMEASE CHAIN REACTION

8/13/2019 PCR 31-08-12

14/44

It was invented in 1983 by Dr. Kary Mullis, for which hereceived the Nobel Prize in Chemistry in 1993.

8/13/2019 PCR 31-08-12

15/44

COMPONENTS

8/13/2019 PCR 31-08-12

16/44

Physical Components

8/13/2019 PCR 31-08-12

17/44

The Reaction

THERMOCYCLER PCR tube

reaction volume of 10 200 l in small reaction tubes0.2 0.5 ml volumes

8/13/2019 PCR 31-08-12

18/44

8/13/2019 PCR 31-08-12

19/44

8/13/2019 PCR 31-08-12

20/44

8/13/2019 PCR 31-08-12

21/44

8/13/2019 PCR 31-08-12

22/44

8/13/2019 PCR 31-08-12

23/44

Initialization step: heating at 94 96 for 1 9 minutes. It is onlrequired for DNA polymerases that require heat activation by hotstart PCR.[10]

Denaturation step: heating the reaction to 94 98 C for 20 3seconds. It causes DNA melting of the DNA template, yielding

single-stranded DNA molecules.Annealing step: The reaction temperature is lowered to 50 65for 20 40 seconds allowing annealing of the primers to the single

stranded DNA template. Typically the annealing temperature isabout 5 degrees Celsius below the Tm of the primers used.

8/13/2019 PCR 31-08-12

24/44

Extension/elongation step: Taq polymerase has its optimumactivity temperature at 75 80 C, and commonly a temperatureof 72 C is used with this enzyme. At this step the DNA

polymerase synthesizes a new DNA strand complementary tothe DNA template strand by adding dNTPs that arecomplementary to the template in 5' to 3' direction,

The extension time depends both on the DNA polymerase usedand on the length of the DNA fragment to be amplified. As a

rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute..

8/13/2019 PCR 31-08-12

25/44

Final elongation: t a temperature of 70 74 C for 5

15 minutes after the last PCR cycle to ensure that anyremaining single-stranded DNA is fully extended.

Final hold: This step at 4 15 C for an indefinite timemay be employed for short-term storage of thereaction.

8/13/2019 PCR 31-08-12

26/44

Mineral Oil

Peltier effect

8/13/2019 PCR 31-08-12

27/44

The PCR process

Expo nent ia l amp l i f icat ion : At every cycle, the amount of product isdoubled (assuming 100% reaction efficiency). The reaction is verysensitive: only minute quantities of DNA need to be present. [13] Leveling off s tage : The reaction slows as the DNA polymerase losesactivity and as consumption of reagents such as dNTPs and primers

causes them to become limiting. Plateau : No more product accumulates due to exhaustion of reagentsand enzyme.

http://en.wikipedia.org/wiki/Polymerase_chain_reactionhttp://en.wikipedia.org/wiki/Polymerase_chain_reaction

8/13/2019 PCR 31-08-12

28/44

Chemical Components

1) Target DNA - contains the sequence to be amplified.

2) Pair of Primers - oligonucleotides that define the sequenceto be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.

4) Thermostable DNA Polymerase - enzyme thatcatalyzes the reaction

5) Mg ++ ions - cofactor of the enzyme

6) Buffer solution maintains pH and ionic strength ofthe reaction solution suitable for the activity of theenzyme

8/13/2019 PCR 31-08-12

29/44

PCR PRIMER

8/13/2019 PCR 31-08-12

30/44

PCR PRIMER

LENGTH

SPECIFICITY 1 additional , 4 times more specific 18-24 NUCLEOTIDE

Tm of primer 54 deg or above

preferably not less than 18

Upper limit is less critical, but for entropic reason

28-35 nucleotides, in case of expected heterogeneity isoformsof a protein

8/13/2019 PCR 31-08-12

31/44

Placement of 3 end of primer (5 -6 bases are critical)

Increase length for a purpose to adding extra nucleotides 5end, especially when PCR products need to be cloned RE site nonspecific sequence

Longer primers difficulty in calculation of Tm (nearest

neighbor calc) Around 20 nucleotide primers its;

Tm = 4(G+C)+2(A+T)

8/13/2019 PCR 31-08-12

32/44

TERMINAL NUCLEOTIDES 3 END NUCLEOTIDE Control mispairing

Primers should not be complementary, mainly at 3 end Amplied primer dimer product

GC CONTENT 50-60%

Matching Tm of the primer pair

8/13/2019 PCR 31-08-12

33/44

NESTED PCR

Used to increase sensetivity and specificity

Two pairs of amplification primers

Second pair of primers anneal to the amplifiedfragment produced by first pair of primers

8/13/2019 PCR 31-08-12

34/44

8/13/2019 PCR 31-08-12

35/44

HHV-6 and HTLV-2

Specific Nested PCR System

EXAMPLE

System target HHV-6 U54 gene

Use of the outer primer set, recognize both the Aand B variants, a fragment 0f 383 base pairs inlength

Two separate sets of primers are used to recognisevarient A and varient B

8/13/2019 PCR 31-08-12

36/44

Primers-NESTED PCR

A control inner primer set, run in parallel, detects the

wild-type sequence. In general, the product of the innerset is small, 120-270 bp.

When selecting nested primer sets, special care must begiven to eliminate potential primer dimers and matches

between members of the inner and outer primer sets.

Some of the software programs for primer design havethe selection of nested primers as an option

8/13/2019 PCR 31-08-12

37/44

PRIMER DESIGNING SOFTWARE

Different algorithm

Tm=2(A+T)+4(G+C)

Tm (C) = 81.5 + 0.41(%GC) - (675/N)

3 end GC

Avoid 3 GGG/ CCC

8/13/2019 PCR 31-08-12

38/44

8/13/2019 PCR 31-08-12

39/44

dNTP

8/13/2019 PCR 31-08-12

40/44

dNTPs The usual dNTP concentration is between 40 M and 200 M o

EACH of the four dNTPs.

Excessive dNTP concentrations can inhibit the PCR preventing theformation of product.

For longer PCR-fragments a higher dNTPs concentration may berequired.

Suboptimal concentration of nucleotides can cause incompleteprimer elongation or premature termination of DNA synthesisduring the elongation step of the PCR cycle.

8/13/2019 PCR 31-08-12

41/44

1 unit of the Taq enzyme should be used for a 25l reaction.Suboptimal concentration of the Taq enzyme can cause incompleteprimer elongation or premature termination of the PCR productsynthesis during the elongation step of a PCR cycle.

Too much Taq will result in an excessive background of unwantedDNA fragments (a smear on a gel) while a huge excess may cause thereaction to fail with no product being detected.

A Taq concentration of depending upon expected amplicationproduct

Taq Pol

8/13/2019 PCR 31-08-12

42/44

Magnesium is a required cofactor for thermostable DNA polymerases.

Mg 2+ in the PCR mixture stabilizes dsDNA and raises the Tm.

Mg 2+concentration therefore is an important for controlling thespecificity of the reaction.

A low Mg 2+ concentration requires more stringent base pairing in theannealing step.

Too few Mg 2+ ions result in a low yield of PCR product; too manyMg 2+ ions increase the yield of non-specific products and promotemisincorporation.

Mg++

KCl

8/13/2019 PCR 31-08-12

43/44

Potassium chloride (KCl) is normally used in a PCR amplificationat a final concentration of 50mM.

To improve the PCR amplification of DNA fragments, especiallyfragments in the size range 100bp to 1000bp, a KCl concentrationof between 70mM and 100mM is sometimes recommended.

For the amplification of longer products a lower salt concentration

appears to be better. But the PCR amplification of short productsworks better at higher salt concentrations.

This is probably because an increase in salt concentration permitsshorter DNA molecules to denature preferentially to longer DNAmolecules. Shorter molecules are therefore amplified better athigher salt concentration.

It should be remembered however that a salt concentration above50mM can inhibit the Taq polymerase.

KCl

8/13/2019 PCR 31-08-12

44/44

Gene Cloning and DNA analysis: An Introduction 6 th ed.T.A. Brown, Blackwell

http://www.highveld.com/pages/pcr-troubleshooting.html