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 .

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AGAROSE GEL ELECTROPHORESIS .

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Preparation of gDNA .

PURIFYING NUCLEIC ACID FRAGMENTS FROM GEL .

POLYMEASE CHAIN REACTION .

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

COMPONENTS .

Physical Components .

5 ml volumes .The Reaction PCR tube THERMOCYCLER reaction volume of 10–200 μl in small reaction tubes 0.2–0.

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Typically the annealing temperature is about 5 degrees Celsius below the Tm of the primers used. yielding single-stranded DNA molecules. Annealing step: The reaction temperature is lowered to 50–65 °C for 20–40 seconds allowing annealing of the primers to the singlestranded DNA template.Initialization step: heating at 94–96 ° for 1–9 minutes. It causes DNA melting of the DNA template. . It is only required for DNA polymerases that require heat activation by hotstart PCR.[10] Denaturation step: heating the reaction to 94–98 °C for 20–30 seconds.

and commonly a temperature of 72 °C is used with this enzyme. the DNA polymerase will polymerize a thousand bases per minute. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. at its optimum temperature. .Extension/elongation step: Taq polymerase has its optimum activity temperature at 75–80 °C. As a rule-of-thumb. 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..

. Final hold: This step at 4–15 °C for an indefinite time may be employed for short-term storage of the reaction.Final elongation: t a temperature of 70–74 °C for 5– 15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

•Mineral Oil •Peltier effect .

Plateau: No more product accumulates due to exhaustion of reagents and enzyme. .[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. The reaction is very sensitive: only minute quantities of DNA need to be present.The PCR process Exponential amplification: At every cycle. the amount of product is doubled (assuming 100% reaction efficiency).

cofactor of the enzyme 6) Buffer solution – maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme . 4) Thermostable DNA Polymerase .contains the sequence to be amplified.oligonucleotides that define the sequence to be amplified.deoxynucleotidetriphosphates: DNA building blocks. 3) dNTPs .Chemical Components 1) Target DNA . 2) Pair of Primers .enzyme that catalyzes the reaction 5) Mg++ ions .

low concentrations of primer.Target DNA  Using too much total DNA results false priming and even poor DNA synthesis due to the obstructed diffusion of largeTaq polymerase molecules.reamplification reaction  When the total amount of the DNA in a PCR reaction is extremely small. When the ratio of the target DNA to the burden DNA is very low. adsorption. and nucleotides are recommended as these generally ensure cleaner product and lower background. there is an increased likelihood of its loss owing to any conceivable cause (clotting. . Taq. target. Ratio of target DNA to burden DNA should be balanced with the number of cycles  However. chemical or enzymatic degradation).

in case of expected heterogeneity – isoforms of a protein . but for entropic reason • 28-35 nucleotides.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.

• Tm = 4(G+C)+2(A+T) . 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.• Placement of 3’ end of primer (5-6 bases are critical) • Increase length for a purpose to adding extra nucleotides – 5’end.

mainly at 3’ end • Amplied primer dimer product GC CONTENT • 50-60% • Matching Tm of the primer pair .TERMINAL NUCLEOTIDES • 3’ END NUCLEOTIDE Control mispairing • Primers should not be complementary.

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 .

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a fragment 0f 383 base pairs in length • Two separate sets of primers are used to recognise varient A and varient B .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.

In general. When selecting nested primer sets. the product of the inner set is small. 120-270 bp. 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 .Primers-NESTED PCR A control inner primer set. run in parallel. detects the wild-type sequence.

(675/N) •3’ end GC •Avoid 3’ GGG/ CCC .5 + 0.PRIMER DESIGNING SOFTWARE Different algorithm •Tm=2(A+T)+4(G+C) •Tm (°C) = 81.41(%GC) .

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

for short target sequences.  In order to generate the required number of PCR product molecules. To amplify short PCR target sequences. concentration of primers higher than 1μM may be necessary.  Therefore. and desirable. 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. a greater number of primers may be needed. if the target fragment length is 100bp. careful calculation of the optimum primer concentration is required. . For example.

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

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.  A Taq concentration of depending upon expected amplication product .Taq Pol  1 unit of the Taq enzyme should be used for a 25μl reaction.  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.

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

. a KCl concentration of between 70mM and 100mM is sometimes recommended. especially fragments in the size range 100bp to 1000bp.  It should be remembered however that a salt concentration above 50mM can inhibit the Taq polymerase.KCl  Potassium chloride (KCl) is normally used in a PCR amplification at a final concentration of 50mM.  For the amplification of longer products a lower salt concentration appears to be better.  To improve the PCR amplification of DNA fragments.  This is probably because an increase in salt concentration permits shorter DNA molecules to denature preferentially to longer DNA molecules. But the PCR amplification of short products works better at higher salt concentrations. Shorter molecules are therefore amplified better at higher salt concentration.

com/pages/pcr-troubleshooting. T.Gene Cloning and DNA analysis: An Introduction 6th ed.highveld.html . Brown.A. Blackwell http://www.