Polymerase Chain Reaction (PCR) and Its Applications

Applications of PCR Detection of pathogens Classification of organisms DNA fingerprinting Drug discovery Genetic matching Genetic engineering Pre-natal diagnosis Classification of organisms Genotyping Molecular archaeology Mutagenesis Mutation detection Sequencing Cancer research

Applications of PCR Basic Research Applied Research Mutation screening Drug discovery Classification of organisms Genotyping Molecular Archaeology Molecular Epidemiology Molecular Ecology Bioinformatics Genomic cloning Site-directed mutagenesis Gene expression studies Genetic matching Detection of pathogens Pre-natal diagnosis DNA fingerprinting Gene therapy

Applications of PCR Molecular Identification Sequencing Genetic Engineering Bioinformatics Genomic cloning Human Genome Project Site-directed mutagenesis Gene expression studies Molecular Archaeology Molecular Epidemiology Molecular Ecology DNA fingerprinting Classification of organisms Genotyping Pre-natal diagnosis Mutation screening Drug discovery Genetic matching Detection of pathogens

MOLECULAR IDENTIFICATION:

Detection of Unknown Mutations Molecular Identification: Detection of Unknown Mutations

SSCP gels: “shifts” representing a mutation in the amplified DNA fragment

Classification of Organisms Molecular Identification: Classification of Organisms Insufficient data 1) Relating to each other 2) Similarities 3) Differences * Fossils * Trace amounts * Small organisms ! DNA !

Rademaker et al. 2001

Detection Of Pathogens Molecular Identification: Detection Of Pathogens

Detection Of Pathogens Molecular Identification: Detection Of Pathogens Sensitivity of detection of PCR-amplified M. tuberculosis DNA. (Kaul et al.1994)

Genotyping by STR markers Molecular Identification: Genotyping by STR markers

Prenatal Diagnosis Chorionic Villus Amniotic Fluid Molecular Identification: Prenatal Diagnosis 644 bp 440 bp 204 bp Chorionic Villus Amniotic Fluid Molecular analysis of a family with an autosomal recessive disease.

SEQUENCING radioactive fluorescent Nucleotides (dNTP) are modified (dideoxynucleotides = ddNTP) NO polymerisation after a dideoxynucleotide! Fragments of DNA differing only by one nucleotide are generated radioactive Nucleotides are either or fluorescent

Classical Sequencing Gel

Reading Classical Sequencing Gels

Sequencing:

Applied Biosystems 310 Genetic Analyzer 6/14/2018 Applied Biosystems 310 Genetic Analyzer

The Process In a Nutshell 6/14/2018 The Process In a Nutshell Amplified DNA samples are injected into a capillary. Fluorescent tags on the DNA fragments are excited by a laser as they pass a window in the capillary, the fluorescence is recorded by a camera, and this signal is converted into a “peak” by the computer software.

6/14/2018 STR data X, Y, X Y

6/14/2018 STR data (cont’d)

6/14/2018 STR data (cont’d) “The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”

Summary blood, chorionic villus, amniotic fluid, semen, hair root, saliva 68,719,476,736 copies Gel Analysis, Restriction Digestion, Sequencing

Conclusion The speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionised molecular biology and made PCR the most widely used and powerful technique with great spectrum of research and diagnostic applications.