Pcr And Types Of Pcr
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PCR AND TYPES OF PCR Submitted to sir Mudassir Submitted by 17049 17048 17028 17036 17040 17051 17038 17035
Bachelors of eastern medicine and surgery
POLYMERASE CHAIN REACTION PCR
It is a molecular technology aim to amplify a single or few copies of DNA to thousands or millions of copies. In vitro technique
HISTORY
The PCR is a technique was Invented by KARY MULLIS in 1983 as a research scientist in California biotech company PCR is now common and often Indespensibel technique used In medical and biological Reasearch labs for a variety of Applications. In 1983 the scientist KARY nobel prize.
MULLIS was awarded the
PCR WHY “ POLYMERASE “ ? BECAUSE THE ONLY ENZYME USED IN THE REACTION IS DNA POLYMERASE. WHY “ CHAIN “ BECAUSE THE PRODUCT OF THE FIRST REACTION BECOME THE SUBSTRATES OF THE FOLLOWING ONE AND SO ON .
With this technique ,small amounts of genetic material can be amplified,to be abel to identify and manipulate DNA ,detect infectious organisms ,detect genetic variations including mutation in human genes and numerous others tasks.
SETTING UP PCR REACTION Constituents of PCR reaction: 1.Target DNA 2. PAIRS OF PRIMERS 3.dNTPs 4.Thermostable DNA Polymerase 5.Mg++ions 6.Buffer sol.
STEPS OF PCR:
1. Denaturation 2.Annealing 3.Extention
TYPES OF PCR: PCR is of different types 1.Inverse PCR 2.Multiplex PCR 3.Nested PCR 4.Colony PCR 5.Real time PCR
1: INVERSE PCR
Also k/a inverted PCR or inside PCR.
It is used to amplify unknown DNA segment that flanks one end of known DNA sequence for which no primers are availabel.
INVERSE PCR STEPS
Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion . Self-ligation is induced under low concentrations causing the phosphate backbone to reform . This gives circular DNA ligation product . Target DNA is then restriction digested with a known endonuclease this generates a cut within the known internal sequence generating a linear prodcut with known terminal sequences.this can know be used for PCR . Standard PCR is conducted with primers complementery to the now known internal sequences.
SIGNIFICANCE
Amplification and identification of sequences flanking transposable elements. Cloning of unknown cDNA sequence from total RNA . Construction of end specific probes for chromosome walking. Amplification of integration sites used by viruses and transgenes.
2:MULTIFLEX-PCR:
It is a special type of the PCR used for detection of multiple pathogens by using multiple primers sets each one targets a particular pathogens.
Uses:
This permits the simultaneous analysis of multipel targets in a single sample.
3:NESTED-PCR:
Two pairs instead of one pair of PCR primers are used to amplify a fragment . First pair amplifies a fragment similar to standard PCR. Second pairs bind inside the 1st PCR product fragment allow amplification of 2nd PCR product which is shorter than 1st one.
USES: Detection of pathogens that occur with very few amount.
4: RT-PCR REVERSE TRANSCRIPTION PCR,REAL TIME PCR:
Used to reverse-transcribe and amplify RNA to cDNA.
PCR is proceded by a reaction using “reverse transcriptase” an enzyme that converts RNA to cDNA. The two reactions may be combined in a tube.
Uses: 1:Detection of RNA virus like HCV 2:Detection Of other M.O. through targeting of their ribosomal RNA.
REVERSE TRANSCRIPTION PCR, REAL TIME – PCR:
5:COLONY PCR:
Bacterial colonies are secreened directly by PCR,for example, the screen for correct DNA-vector constructs. Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix.
APPLICATIONS OF PCR :
1:Molecular Identification: Molecular archaeology Molecular Epidemiology Molecular Ecology DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens
2:SEQUENCING Bioinformatics Genomic coloning Human genome project
3:GENETIC ENGINEERING Site-detecting mutagenesis Gene expression studies.
Bachelors of eastern medicine and surgery
POLYMERASE CHAIN REACTION PCR
It is a molecular technology aim to amplify a single or few copies of DNA to thousands or millions of copies. In vitro technique
HISTORY
The PCR is a technique was Invented by KARY MULLIS in 1983 as a research scientist in California biotech company PCR is now common and often Indespensibel technique used In medical and biological Reasearch labs for a variety of Applications. In 1983 the scientist KARY nobel prize.
MULLIS was awarded the
PCR WHY “ POLYMERASE “ ? BECAUSE THE ONLY ENZYME USED IN THE REACTION IS DNA POLYMERASE. WHY “ CHAIN “ BECAUSE THE PRODUCT OF THE FIRST REACTION BECOME THE SUBSTRATES OF THE FOLLOWING ONE AND SO ON .
With this technique ,small amounts of genetic material can be amplified,to be abel to identify and manipulate DNA ,detect infectious organisms ,detect genetic variations including mutation in human genes and numerous others tasks.
SETTING UP PCR REACTION Constituents of PCR reaction: 1.Target DNA 2. PAIRS OF PRIMERS 3.dNTPs 4.Thermostable DNA Polymerase 5.Mg++ions 6.Buffer sol.
STEPS OF PCR:
1. Denaturation 2.Annealing 3.Extention
TYPES OF PCR: PCR is of different types 1.Inverse PCR 2.Multiplex PCR 3.Nested PCR 4.Colony PCR 5.Real time PCR
1: INVERSE PCR
Also k/a inverted PCR or inside PCR.
It is used to amplify unknown DNA segment that flanks one end of known DNA sequence for which no primers are availabel.
INVERSE PCR STEPS
Target DNA is lightly cut into smaller fragments of several kilobases by restriction endonuclease digestion . Self-ligation is induced under low concentrations causing the phosphate backbone to reform . This gives circular DNA ligation product . Target DNA is then restriction digested with a known endonuclease this generates a cut within the known internal sequence generating a linear prodcut with known terminal sequences.this can know be used for PCR . Standard PCR is conducted with primers complementery to the now known internal sequences.
SIGNIFICANCE
Amplification and identification of sequences flanking transposable elements. Cloning of unknown cDNA sequence from total RNA . Construction of end specific probes for chromosome walking. Amplification of integration sites used by viruses and transgenes.
2:MULTIFLEX-PCR:
It is a special type of the PCR used for detection of multiple pathogens by using multiple primers sets each one targets a particular pathogens.
Uses:
This permits the simultaneous analysis of multipel targets in a single sample.
3:NESTED-PCR:
Two pairs instead of one pair of PCR primers are used to amplify a fragment . First pair amplifies a fragment similar to standard PCR. Second pairs bind inside the 1st PCR product fragment allow amplification of 2nd PCR product which is shorter than 1st one.
USES: Detection of pathogens that occur with very few amount.
4: RT-PCR REVERSE TRANSCRIPTION PCR,REAL TIME PCR:
Used to reverse-transcribe and amplify RNA to cDNA.
PCR is proceded by a reaction using “reverse transcriptase” an enzyme that converts RNA to cDNA. The two reactions may be combined in a tube.
Uses: 1:Detection of RNA virus like HCV 2:Detection Of other M.O. through targeting of their ribosomal RNA.
REVERSE TRANSCRIPTION PCR, REAL TIME – PCR:
5:COLONY PCR:
Bacterial colonies are secreened directly by PCR,for example, the screen for correct DNA-vector constructs. Colonies are sampled with a sterile pipette tip and a small quantity of cells transferred into a PCR mix.
APPLICATIONS OF PCR :
1:Molecular Identification: Molecular archaeology Molecular Epidemiology Molecular Ecology DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens
2:SEQUENCING Bioinformatics Genomic coloning Human genome project
3:GENETIC ENGINEERING Site-detecting mutagenesis Gene expression studies.
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