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Outline

INTRODUCTION TO MOLECULAR BIOLOGY PCR

PCR Definition and Perspective Polymerase chain reaction (PCR) is a rapid in vitro procedure for enzymatic amplification of specific segments of DNA, typically in the range of 100-300 bp long. PCR allows rapid replication of a single copy of DNA into millions of copies. For example, starting with about 100 ngm of genomic mammalian DNA (~104 molecules), the standard PCR protocol yields an easily visible product on an ethidium bromide stained gel. For this reason, PCR is one of the widely used techniques in modern medicine and has had a great impact in every aspect of modern molecular biology.

PCR Applications      PCR is used in the following conditions: Assays for the presence of infectious agents Prenatal diagnosis of genetic diseases Direct cloning from genomic DNA or cDNA Quantitation of rare DNA by PCR Amplification of RNA by PCR (RT-PCR) In vitro mutagenesis and engineering of DNA Genetic fingerprinting of forensic samples Analysis of allelic sequence variations

PCR Applications Analysis of RNA transcript structure Ligation-mediated PCR for genomic sequencing and footprinting Direct nucleotide sequencing of genomic DNA or cDNA Direct sequencing of PCR products Preparation of ss products for dideoxy sequencing Preparation of ds products for dideoxy sequencing Labeling products for chemical sequencing Preparation of ss and ds products for cycle sequencing and automated sequencing

PCR Steps       The PCR consists of repetition of the following three steps. Denaturation: in this step the two strands of the DNA are separated. This is normally done at 94 ° C. Annealing: in this step the primers bind to the DNA. This is normally achieved at 55° C Extension: in this phase, using the DNA template, the polymerase adds the dNTPs to the primer and causes the extension of the primer. This is normally done at 72° C

PCR Steps The denaturation, annealing and extension steps are then repeated in succession for 25-35 cycles, hence the term polymerase chain reaction.

PCR Steps The amplified products are then evaluated by the following means: The products are subjected to gel electrophoresis and are viewed by staining with ethidium bromide. This allows determining the size of the amplified products. The size of the amplified product should correspond to the expected size of the band. The identity of the amplified products are verified by restriction enyme digestion, sequencing and/or Southern blotting.

PCR Components DNA fragment to be amplified Two single stranded oligonucleotide primers flanking the DNA segment DNA polymerase Deoxyribonucleoside triphosphates (dTNPs) Buffer Salts Thermocycler       After addition of the PCR components the microfuge is layered with mineral oil to prevent evaporation.

PCR Optimization of the procedure       Each PCR reaction should be optimized. The following parameters play a major role in insuring that the PCR works: Purity and amount of the template Concentration of MgCI2 Reagent purity Nuclease contamination DNA contamination Appropriately designed primers Taq DNA Polymerase Concentration Activity Heat stability

PCR Primer design and selection Primers should be able to hybridize specifically to the sequence of interest

PCR Primer design

PCR Optimization of the procedure Concentration of deoxyribonucleoside Triphosphates (200 mM) Thermocycling Parameters Denaturation temperature Annealing temperature Extension "Ramp time"

PCR Primer design and selection The size of the amplified product should be optimal (~ 300 bp) Appropriate primer length (20-30 bases; 16 bases if found once in human genome)  Appropriate primer sequence GC content similar to template Avoiding stretches of polypurines or polypyrimidines Use of computer prediction programs Avoiding self complementarity to prevent "Primer-Dimer formation”

PCR Primer design and selection Avoiding hairpin loop formation The 3’ ends of the primer should not complement non-specific targets such as  vector, cDNA insert, genomic DNA or PCR product The melting points of both primers should be similar. The 3’ terminal nucleotides of primers should end in a G or C, or the same base. Primers should have a moderate GC content.

PCR Primer design and selection Primers should have a moderate GC content. A run of >6 G’s or A’s should be avoided AAG at 3’ end should be avoided GGAG at 3’ end should be avoided

PCR Enzymes

DIFFERENTIAL DISPLAY OF mRNA BY PCR