PCR & its Applications

Assignment

Presentation To :Dr. Sunita Khatak

Presented by:Sandhya Kaushik(2508463)

27-11-2010

Polymerase Chain Reaction Technique

Developed By Kary Mullis in 1985. Selective amplificatiion of a chosen region of a DNA molecule. It generates microgram copies of the desired DNA or RNA segment, present even as a single copy in the initial prepration.

Utilization for PCR

Target Secquence Two nucleotide Primers Four Deoxynucleoside Triphosphate as TTP(thymidine Tryphosphate), dCTP(deoxycyctidine triphosphate) dATP(deoxyadenosine triphosphates) TTP(Thymidine triphosphate) A heat stable DNA polymerase

Procedure for PCR

Denaturation Annealing Primer Extension

Denaturation
Temperature between 90-98C. First Cycle of PCR For 2 minutes

Annealing
At temperature 40-60C It permits that the annealing of the primer to the complimentary secquence in the DNA. At 3 prime end For 1 minute

Primer Extension
DNA polymerase Systhesizes the complementary strands by utilizing 3 prime OH of the Primer. At temperature 72 C.

PCR Machine

PCR Primer
Should corresponding to the target region. Size about 20 bp long. Should not in same direction. Due to hybridization of one or both of the primers to non target sites on the template DNA molecule undesired amplification products.

Annealing Temperature
Very important since the success and specificity of PCR depend on it. Because DNA-DNA hybridization is a temp dependent phenomena. If high paring does not take place, PCR will fail. If low temp. not stably pair. Specificity of PCR describe the probability of a non target sequence being amplified ;the lower this probability, the higher is the PCR specificity.

Cont.
Ideal Annealing Temp. must be low enough to enable hybridization b/w primer and template but high enough to prevent amplification of non target site. IAT usually 1-2C lower than melting temp. Melting Temp. Tm=[4(G+C)]+[2(A+T)] the two primer for a PCR must be design such a way that they both have an identical Tm.

PCR Efficiency
The efficiency of amplification decline with an increase in the length of target sequence. Efficiency of PCR is effected by primer length; it decline if the primer used for PCR are too long Annealing temperature has a marked influence on PCR efficiency; a temp. higher than the ideal annealing temp reduces PCR afficiency. Primer sequence; dimer Target sequence; GC rich may form secondary structure in the single strand produces by denaturation, it could reduces PCR efficiency. Addition of certain proteins BSA enhance PCR efficiency by protecting the DNA polymerase by binding to PCR inhibitors.

Variations of PCR
PCR is highly versatile technique and have a no. types. 1.Inverse PCR 2.Anchored PCR 3.RT-PCR 4.Asymmetric PCR 5.Nested PCR

Nested PCR
Target sequence is amplified a pair of PCR primer. A portion of amplification product is re amplified using another pair of PCR primer complementary to the region of first pair of primer.

Nested PCR

Inverse PCR
Inverse PCR is used for the synthesis of unknown region which are either side on the known region. This is done by help of primer Oligonucliotide complementary to the 5 prime end. Cut the target sequence by R.E. Restriction fragment is circularized and ligated low concentration of DNA. Exponentially amplified of unknown region.

Applications of PCR
PCR can be used to amplify specific gene present in different individual of a species even in diff. gamtes or somatic cell say humans sperm of an individual. This copies can be used for cloning. PCR has been used to study DNA polymorphism in genome of random sequence as primer for example RAPD which is detected band after electroforces. PCR can be used to detect the presence of a gene tranfered into organism. Amplification occur only when trans gene present in the organism. Detection can be done by using nucleic acid hybridization (colony, colony blot, southren, northren hybridization). These approches are expensive and take longer time and used raidoactivity but PCR detection take a single day and not used radioactivity.

 Microdissected segment of chromosome for example salivary gland of chromosome can be used for PCR amplification to determined the physical location of gene in chromosome. PCR can be used to generate single strand used for DNA sequencing, thermal cycle sequencing. PCR can be used to produced cDNA copies of mRNA this is don by Reverse Transcriptase PCR and million of copies of DNA duplex is done by Reverse Transcriptase PCR. DNA fingerprinting is now almost exclusively based on PCR. PCR is finding an increasing application of toxonomy example is microbial toxonomy. PCR can be used to prenatal diagnosis of genetic diseases i.e sickle cell anemia. Reverse Trancriptase PCR can also provise information on the activity of tumer cell and virus.

Cont