Polymerase chain reaciton

1. Polymerase chain reaciton An Introduction

2. 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

3. Revolutionized many areas of genetic research including • Genetic disease diagnosis • Forensic medicine • Molecular evolution • Production of therapeutic proteins • MORE

4. 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

5. 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

6. 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

7. 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

8. 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

9. 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

10. 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

11. PerformingPCR • The PCR mixture is taken through several replication cycles

12. 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

13. 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!!

14. 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

15. 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

16. 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

17. 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

18. 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 polymerase X 52 • ------ 2ul forward primer X 52 • ------- 2ul reverse primer X 52 • ------ 2ul PCR buffer with MgCl2 usually a 10x buffer X52 • ------ 2ul dNTP’s X 52 • ------ 9.9 ul water X 52 • Vt 20ul 1040ul

19. 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)

20. The level of investigation • Karyotype………………………………Sequencing • Entire genome………………………….Actual base pairs PCR—level of the Gene

21. Karyotyping Turner’s Down’s

22. Sequencing provides Base Pair Info ddNTP with radioactive label on Acrylamide gel ddNTP with fluorescent tag Eluded from acrylamide gel

23. DNA Fingerprinting • Procedure may use different techniques • RFLP’s -- Restriction fragment length polymorphism • VNTR’s –Variable Number of Tandem Repeat

24. DNA Fingerprinting A SNP produces a change in a restriction enzyme site Changes in nucleotide sequence can be detected

25. PCR-RFLP For disease diagnosis Identification of infection

26. 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

27. 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

28. 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.

29. 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

30. 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.

31. 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.

32. 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.

33. VNTR’s Can be Used to Create Genetic Fingerprint

34. 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