1. DNA extraction, RE digestion Procedure What PCR Amplification of specific segment of DNA in-vitro 1.

Denaturation 95oC Double stranded single stranded 2. Primer annealing 64 oC DNA primer anneals to complementary DNA strand, Hbonds formation, complementary base pairing 3. Chain Extension 72 oC Taq polymerase works optimally for chain extension, forming phosphodiester bonds Gel electrophoresis Analyse and verify DNA fragments ( PCR products) separate segments based on size 1. subject DNA under electric field DNA vely charged sugar phosphate backbone movement towards +ve electrode anode when subjected to electric field 2. Agarose with wells Polysaccharide Meshwork of polymer fibres, impedes movement of DNA fragment longer fragments migrate slower, smaller fragments migrate faster 3. DNA with dense loading buffer Glycerol: sinks DNA to bottom of well Loading dye (tracking dye) 4. Visualing DNA Stain gel with DNA binding dye Ethidium bromide fluoresce under UV Nucleic acid hybridization southern blotting Identify which DNA fragments contain target region 1. Place nitrocellulose membrane over agarose gel 2. Alkaline solution Denature double stranded DNA to single stranded 3. Nucleic acid hybridization: add specific single stranded radioactively labeled DNA probe binds to specific complementary target region hybridisation: complementary base pairing occurs 4. Wash off unhybridised probes 5. Autoradiography 6. Pick out specific bands and nucleotide sequences

Method

*3rd seq: pure target region PCR tube Buffer with Mg2+: cofactor Free nucleotides dNTP Advantage Sensitivity Speed

Limitations

Taq polymerase lacks 3 to 5 proof reading ability Mistakes compounded, cannot exceed 30 cycles (25 norm) Requires K of sequences flanking target region to be amplified Limit to size of DNA fragment (3kb) Polymerase falls of template, continue chain extension Exponential amplication of contaminant DNA

Many different segments(size differ by small amount) close together smears on gel electrophoresis instead of distinct bands

Applications PCR products: gene fragments blunt ends >design primers to include ecoR1 restriction sites /add linkers flanking regions >cut with enzymes, ligate into plasmid recombinant plasmid >transformation, transcribed, translated

Detect and Analyze Restriction fragment length polymorphism Individuals have unique restriction patterns Repeat diff: diff no. of short tandem repeats (STR) and variable no. tandem repeats (VNTR) - DNA finger printing: Use a few different probes for different vNTR compare multiple polymorphic loci nucleotide difference: single nucleotide polymorphism - Disease detection:Direct method SNP occurring at coding region alter DNA seq recognized by restriction enzyme Due to snp, enzymes recognise mutated region loss of restriction sitelarger gene fragment - Comparison of different restriction patterns identifies indiv carrying disease allele - Indirect method: SNP lie in non-coding region e.g. PFK: polymorphic diff lies in region flanking gene - Some disease caused by 1 possible mutations: - Possible crossing over between polymorphic locus and disease causing mutations Genomic mapping: building a picture of arrangement Recombination frequency: 1% RF = 1 m.p.