Polymerase chain reaciton
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Polymerase chain reaciton. An Introduction. Polymerase chain reaction or PCR is a powerful technique allowing scientists to amplify a specific DNA sequence millions of times in just a few hours

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Polymerase chain reaciton

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Polymerase chain reaciton

Polymerase chain reaciton

An Introduction


Polymerase chain reaciton

  • Polymerase chain reaction or PCR is a powerful technique allowing scientists to amplify a specific DNA sequence millions of times in just a few hours

  • It was invented by Dr. Kary Mullis in 1983 and in 1993 he received the Nobel Prize in Chemistry for this work


Polymerase chain reaciton

  • Revolutionized many areas of genetic research including

  • Genetic disease diagnosis

  • Forensic medicine

  • Molecular evolution

  • Production of therapeutic proteins

  • MORE


Basis of pcr lies in dna replication

Basis of PCR lies in DNA replication

  • Within a dividing cell, DNA replication involves a series of enzyme-mediated reactions

  • The result is a faithful copy of the entire genome


Cellular dna replication

Cellular DNA replication

  • Enzymes first unwind the DNA double helix into single strands

  • RNA polymerase synthesizes a short stretch of RNA

  • This is complementary to one of the DNA strands at the start site of replication


Cellular dna replication1

Cellular DNA replication

  • The RNA/DNA heteroduplex is a “PRIMING SITE” for the attachment of the DNA polymerase

  • DNA Polymerase produces a complementary DNA strand by adding nucelotides in the 5’ to 3’ direction


Pcr mimics dna replication in a test tube

PCR mimics DNA replication in a test tube

  • During PCR, high temperature is used to separate the DNA molecules into single strands

  • The DNA duplex is held together by hydrogen bonds which are easily disrupted—heating the sample to high temperatures approaching the boiling point of water


Pcr mimics cellular dna replication

PCR mimics Cellular DNA replication

  • Once separated, synthetic sequences of single-stranded DNA serve as primers.

  • These primers can easily be made and purchased

  • Generally 20-30 nucleotides long

  • They will bracket the DNA segment to be copied


Pcr primers

PCR Primers

  • One primer is complementary to one DNA strand at the beginning of the target region

    A T C G C G G T A T C G T G T C A T G C T G T T G C A G A T C

    A A C G T C A T C

    A T C G C G G T

  • T A G C G C C A T A G C A C A G T A C G A C A A G C T C T A G

  • A second primer is complementary to the other strand at the end of the target region


Performing pcr

Performing PCR

A small quantity of target DNA is added to a test tube

This tube will have a buffered solution containing

  • DNA polymerase (Taq Polymerase)

  • Short oligonucleotides primers

  • Four deoxnucleotides and MgCl2


Performingpcr

PerformingPCR

  • The PCR mixture is taken through several replication cycles


Thermal cycling

Thermal Cycling

  • One to several minutes at 94-96oC—DNA denatures—the chains come apart

  • One to several minutes at 50-65oC --primers find complementary bases and pair (hybridize or “anneal”)

  • One to several minutes at 72oC during which DNA polymerase binds and extends a complementary strand from each primer


Polymerase chain reaciton

  • As amplification proceeds, the DNA sequence between the primers doubles after each cycle.

  • Following thirty such cycles, a theoretical amplification factor of one billion is reached!!


Pcr automation due to 2 inventions

PCR automation due to 2-inventions

First…..

  • Discovery of Thermus Aquaticus—a heat stable DNA polymerase found in bacteria that live in hot springs

  • Called “Taq” DNA polymerase

  • Remains active in repeated heatings


Pcr automation due to 2 inventions1

PCR automation due to 2-inventions

  • Second , DNA thermal cyclers have been invented in which a computer controls the repetitive temperature changes required for PCR


Challenges in pcr technology

Challenges in PCR Technology

  • The process of PCR is simple but difficulties are often encountered

  • These include:

    • DNA samples are often imperfect due to contamination or mishandled

    • PCR inhibitors are present in cells and common buffers

      • DNAses/RNAses/TE

  • Contamination of workspace/ pipets/technician contamination


Challenges in pcr technology1

Challenges in PCR Technology

  • Many PCR protocols will need optimization

    • The cycling temperatures

      • Base content of primers

      • Length of primers

  • Concentration of MgCl2

  • Starting concentration usually 25mM

  • Serial dilution to determine optimal concentration with 1mM /10mM common


Standardizing a technique

Standardizing a technique

  • Optimized protocol is often scaled up for several samples

  • PCR master mix prepared

    • Example---50 sample must be tested for a specific gene

    • Each tube ------ 2ul DNA Varies from each sample

      • ------ 0.1ul Taq polymeraseX 52

      • ------ 2ul forward primerX 52

      • ------- 2ul reverse primerX 52

      • ------ 2ul PCR buffer with MgCl2 usually a 10x buffer X52

      • ------ 2ul dNTP’sX 52

      • ------ 9.9 ul waterX 52

      • Vt 20ul 1040ul


Application of pcr technology

Application of PCR Technology

  • PCR is used commonly for

    • Solving crimes (forensics/ criminology)

    • Identifying persons (missing children, soldiers)

    • Establishing relationships (paternity)

    • Medical diagnostics (presence of specific genes)

    • Therapeutic drug design

    • Evolutionary studies (relationships/genetic similarities between classes of organisms—current and preexisting)


The level of investigation

The level of investigation

  • Karyotype………………………………Sequencing

  • Entire genome………………………….Actual base pairs

    PCR—level of the Gene


Karyotyping

Karyotyping

Turner’s

Down’s


Sequencing provides base pair info

Sequencing provides Base Pair Info

ddNTP with radioactive label on Acrylamide gel

ddNTP with fluorescent tag

Eluded from acrylamide gel


Dna fingerprinting

DNA Fingerprinting

  • Procedure may use different techniques

    • RFLP’s -- Restriction fragment length polymorphism

    • VNTR’s –Variable Number of Tandem Repeat


Dna fingerprinting1

DNA Fingerprinting

A SNP produces a change in a restriction enzyme site

Changes in nucleotide sequence can be detected


Pcr rflp

PCR-RFLP

For disease diagnosis

Identification of infection


Dna fingerprinting with vntr s

DNA Fingerprinting with VNTR’s

  • VNTR = Variable Number of Tandem Repeats

  • Regions on a chromosome where a specific sequence is repeated, one after another.

  • The number of repeats varies from each person

  • Typically each person will have a different number on the maternal and paternal chormosome


Vntr s

VNTR’s

  • VNTR’s

    • Vary in size (9-80 nt)

    • Copied by PCR to produce fragments that vary in size

    • Primers are created that sit outside the repeated area

    • Identified using electrophoresis


Vntr and disease

VNTR and Disease

  • Trinucleotide, or triplet repeats, consist of three consecutive nucleotides that are repeated within a region of DNA (for example, CCG CCG CCG CCG CCG). These are found in the genome of humans and many other species. All possible combinations of nucleotides are known to exist as triplet repeats, though some, including CGG and CAG, are more common than others.


Vntr and disease1

VNTR and Disease

  • Diseases with Triplet Repeats in Noncoding Regions

  • Fragile X syndrome (CGG repeat) Fragile XE syndrome (GCC repeat) Friedreich ataxia (GAA repeat) Myotonic dystrophy (CTG repeat) Spinocerebellar ataxia type 8 (CTG repeat) Spinocerebellar ataxia type 12 (CAG repeat)

  • Diseases with (CAG)n Repeats in Coding Regions

  • Spinobulbar muscular atrophy (Kennedy's disease)Huntington's diseaseDentatorubral-pallidoluysian atrophySpinocerebellar ataxia types 1, 2, 3, 6 and 7


Disease with vntr

Disease with VNTR

  • John Todd's group at the University of Cambridge, in tandem with colleagues in Florida and Canada, confirmed a type 1 diabetes-associated polymorphism to be a region of DNA, close to the insulin gene, called a VNTR (variable number of tandem repeats). As its name suggests, this region is made up of a small sequence of DNA repeated many times.


Vntr and disease2

VNTR And Disease

  • In Europeans, there are two main categories of VNTR alleles: those with fewer than 50 repeats, and those with more than 200 repeats. Studies have shown that people with only the low number repeat are more likely to have type 1 diabetes than those with at least one higher number allele.


Vntr and disease3

VNTR and Disease

  • “Carrying a protective allele gives you at least 50 per cent protection from the disease," says Professor Todd. "It's a rather dramatic effect."

  • The variability in the length of the repeat is important in the way autoimmunity arises or is prevented. The VNTR plays a role in regulating the expression of the insulin gene in the thymus, a gland behind the breastbone in which the immune system 'learns' to distinguish foreign matter from self.


Vntr s can be used to create genetic fingerprint

VNTR’s Can be Used to Create Genetic Fingerprint


Forensics

Forensics

  • Crime scene analysis requires an understanding and application of

    • Biology

    • Chemistry

    • Physics

    • Mathematics,

    • Sociology

      Tools include

      DNA, hair, materials (fabric,glass, metal, etc) force analysis (splatter marks, skid marks , etc) and many more physical, chemical and biological


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