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 MULLIS was awarded the
nobel prize.
 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.