Plan - San Diego Miramar Collegefaculty.sdmiramar.edu/bhaidar/bhaidar 210A web uploads/Bio...

PlanA. Link the technique of PCR to the basic knowledge about DNA replication

a. Review DNA replication in vivob. DNA replication in vitroc. Compare and contrast the in vivo and in vitro DNA replication

B. PCR as a technique for selective amplification of specific segments of DNA from a complex genomea. DNA replication cycles exponential amplification with doubling b. Steps of each cycle of replication

C. Important considerations for preparation of Genomic DNA from cheek cells for usea. Components of cell lysis solution and consideration of Mg+2 (Chelex)b. PCR reaction components

PCRBackground Information

• Review DNA replication in vivo • DNA replication in vitro• Compare in vivo & in vitro DNA

synthesis• Genomic DNA and cell lysis• Components of lysis mixture• PCR

• Steps • Reaction mixture components

• Human genomic DNA to be amplified (next lab)

Basic Lab Skills

• Genomic DNA preparation• Setting-up a PCR reaction• Analysis using DNA Gel

Electrophoresis (next lab)

Compare and contrast DNA Replication in vivo

PCR-DNA Amplification in vitro

DNA Replication • Initiation

Strand Separation by helicasePrimer synthesis by primase

• ElongationExtension and polymerization of nucleotides by DNA polymerase using dNTPs: dATP, dTTP, dCTP, and dGTP

• TerminationLeading strand- End of template strandLagging strand- Replace RNA primer with DNA strand & fill-in gaps with DNA ligase

Comparison of In vivo and In vitro reactions

In vivo In vitro

Strand separation –

Priming –

Elongation –

Preparation of genomic DNA

• Lyse or break cells by disrupting cell membranes using a detergent

• Digest with a protease to release naked DNA

• Separate DNA from digested proteins

PCR: polymerase Chain ReactionDNA amplification in a test tube

http://www.sumanasinc.com/webcontent/anisamples/molecularbiology/pcr.html

http://www.dnalc.org/ddnalc/resources/animations.html

PCR animation Dolan learning Center• http://www.dnalc.org/

• http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Polymerase Chain Reaction (PCR)http://www.biology.arizona.edu/

Target region of PCR amplificationAn Alu repeat Element inserted in intron 8 of the Tissue PlasminogenActivator (TPA gene) in some individuals

Position of PCR primers

Position of PCR primers

Individual genotypes

• cDNAhttp://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

• Plasmid cloning and transformationhttp://www.sumanasinc.com/webcontent/anisamples/molecularbiology/plasmidcloning_fla.html

• Steps of gene cloning http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#

Plasmid Cloning