Morgan 1922 : 2000 genes in 4 chromosome of D.

melanogaster

Avery, MacLeod & McCarty 1944 DNA is the genetic material

Harsey Chase 1952 DNA is the genetic material Delbruck, Chargaff, Crick, Monod DNA stracture

Recombinant DNA technology / genetic Engineering

Gene cloning
DNA sequencing

PCR

Kary Mullis 1985

Coast of California drive

PCR

DNA in a Cell

AGAROSE GEL ELECTROPHORESIS

Preparation of gDNA

PURIFYING NUCLEIC ACID FRAGMENTS FROM GEL

POLYMEASE CHAIN REACTION

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

COMPONENTS

Physical Components

The Reaction

PCR tube

THERMOCYCLER

reaction volume of 10200 l in small reaction tubes 0.20.5 ml volumes

Initialization step: heating at 9496 for 19 minutes. It is only required for DNA polymerases that require heat activation by hotstart PCR.[10] Denaturation step: heating the reaction to 9498 C for 2030 seconds. It causes DNA melting of the DNA template, yielding single-stranded DNA molecules.

Annealing step: The reaction temperature is lowered to 5065 C for 2040 seconds allowing annealing of the primers to the singlestranded DNA template. Typically the annealing temperature is about 5 degrees Celsius below the Tm of the primers used.

Extension/elongation step: Taq polymerase has its optimum activity temperature at 7580 C, and commonly a temperature of 72 C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, The extension time depends both on the DNA polymerase used and 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..

Final elongation: t a temperature of 7074 C for 5 15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 415 C for an indefinite time may be employed for short-term storage of the reaction.

Mineral Oil Peltier effect

The PCR process Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very sensitive: only minute quantities of DNA need to be present.[13] Leveling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting. Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

Chemical Components
1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified. 3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks. 4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction

5) Mg++ ions - cofactor of the enzyme

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

Target DNA
Using too much total DNA results false priming and even poor DNA synthesis due to the obstructed diffusion of largeTaq polymerase molecules. Ratio of target DNA to burden DNA should be balanced with the number of cycles However, low concentrations of primer, target, Taq, and nucleotides are recommended as these generally ensure cleaner product and lower background. When the ratio of the target DNA to the burden DNA is very low- reamplification reaction

When the total amount of the DNA in a PCR reaction is extremely small, there is an increased likelihood of its loss owing to any conceivable cause (clotting, adsorption, chemical or enzymatic degradation).

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 isoforms of a protein

 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)

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

NESTED PCR
Used to increase sensetivity and specificity
Two pairs of amplification primers Second pair of primers anneal to the amplified fragment produced by first pair of primers

EXAMPLE
HHV-6 and HTLV-2

Specific Nested PCR System

System target HHV-6 U54 gene

Use of the outer primer set, recognize both the A and B variants, a fragment 0f 383 base pairs in length Two separate sets of primers are used to recognise varient A and varient B

Primers-NESTED PCR
A control inner primer set, run in parallel, detects the wild-type sequence. In general, the product of the inner set is small, 120-270 bp. When selecting nested primer sets, special care must be given to eliminate potential primer dimers and matches between members of the inner and outer primer sets. Some of the software programs for primer design have the selection of nested primers as an option

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

Primers concentration
If the primers are capable of forming dimers, raising their concentration only results in the creation of primer-dimers and does not improve the amplification of the desired PCR product. Primerderived oligomers will possibly contaminate the reaction. If the primers do not form primer-dimers, it is likely that raising the primer concentration will lead to non-specific primer binding and the creation of spurious, undesirable PCR products.

 To amplify short PCR target sequences, careful calculation of the optimum primer concentration is required. For example, if the target fragment length is 100bp, a greater number of PCR product molecules is required to provide a specified amount of amplified DNA (in nanograms) than for a larger target fragment. In order to generate the required number of PCR product molecules, a greater number of primers may be needed. Therefore, concentration of primers higher than 1M may be necessary, and desirable, for short target sequences.

dNTPs
The usual dNTP concentration is between 40M and 200M of EACH of the four dNTPs. Excessive dNTP concentrations can inhibit the PCR preventing the formation of product. For longer PCR-fragments a higher dNTPs concentration may be required. Suboptimal concentration of nucleotides can cause incomplete primer elongation or premature termination of DNA synthesis during the elongation step of the PCR cycle.

Taq Pol
1 unit of the Taq enzyme should be used for a 25l reaction. Suboptimal concentration of the Taq enzyme can cause incomplete primer elongation or premature termination of the PCR product synthesis during the elongation step of a PCR cycle. Too much Taq will result in an excessive background of unwanted DNA fragments (a smear on a gel) while a huge excess may cause the reaction to fail with no product being detected. A Taq concentration of depending upon expected amplication product

Mg++
Magnesium is a required cofactor for thermostable DNA polymerases. Mg2+ in the PCR mixture stabilizes dsDNA and raises the Tm. Mg2+concentration therefore is an important for controlling the specificity of the reaction. A low Mg2+ concentration requires more stringent base pairing in the annealing step. Too few Mg2+ ions result in a low yield of PCR product; too many Mg2+ ions increase the yield of non-specific products and promote misincorporation.

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

To improve the PCR amplification of DNA fragments, especially fragments in the size range 100bp to 1000bp, a KCl concentration of 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 products works better at higher salt concentrations.
This is probably because an increase in salt concentration permits shorter DNA molecules to denature preferentially to longer DNA molecules. Shorter molecules are therefore amplified better at higher salt concentration.

It should be remembered however that a salt concentration above 50mM can inhibit the Taq polymerase.

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

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