structura adn si pcr

Lecture 1 Problem: From an E. coli cell extract, you assay enzyme activity for beta-galactosidase. You divide the extract into two samples, one of which you treat with SDS (sodum dodecyl sulfate). Both samples are further divided into 2 samples each which are alternatively assayed for enzyme activity and subjected to Western Analysis (immunological testing with beta-galactosidase antibody). These are the results:Enzyme Activity Antigenic ResponseExtract only YESYESExtract +SDS NOYES

Give a molecular/biochemical explanation of these results.

Copyright (c) by W. H. Freeman and Company

Lecture 2DNA Structure and ReplicationTopics: StructureSynthesisDNA Sequencing & PCR

Reading: Chapter 4: 101-6; 131-7Chapter 9: 372-5Molecular Biology syllabus web site


All nucleotides have a common structure


There are five principal bases in nucleic acidsA, G, T, C are present in DNAA, G, U, C are present in RNA


Nucleotide subunits are linked together by phosphodiester bonds


Native DNA is a double helix of complementary antiparallel chains held together by: Hydrogen bonding between complementary base pairs (A-T or G-C) Hydrophobic interactions between planar bases


Forces that maintain DNA as a double strand.

are destroyed by formamide, high pH (NaOH), high temperature

H-bondingHydrophobic interactions (cooperative base stacking)


DNA can undergo reversible strand separation


Analysis of DNA denaturationTm= temperature at which half the bases in a double stranded DNA sample have denatured


Many DNA molecules are circular and local unwinding of circular DNA can produce supercoilingsupercoiledrelaxed


RequirementsEnzyme: DNA PolymeraseDNA Template3 OH (primer of DNA or RNA)Deoxynucleoside triphosphates: dATP, dGTP, dCTP, dTTPSynthesis is 5 to 3DNA Synthesis


DNA Synthesis


Features of DNA Polymerasesactivity functionpolymerase synthesis


The growing replication fork shows that both strands are synthesized simultaneously


-Problem- Q: If DNA can only be synthesized in a 5 to 3 direction, and both strands are simultaneously replicated, how can this occur? A: Discontinuous DNA Replication Discontinuous DNA Replication Discontinuous DNA Replication 3553growing fork3553?


The Solution


Synthesis of the lagging strand


DNA Replication Animation


DNA Sequencing with dye terminators


3 OH can be used for phosphodiester bondNo 3 OH: DNA synthesis terminatesIn both cases, DNA polymerase will incorporate nucleoside monophosphates, but..


DNA sequencing: the Sanger (dideoxy) method


Automated DNA sequencing involves use of four different fluorescent primers allowing the simultaneous detection of all four reactions in one sample.


DNA Sequencing Animation


Polymerase Chain Reaction (PCR)


PCR Animation


structura adn si pcr

Documents

denaturedcopyright c

companycopyright c

rna copyright c

dna structure

dna molecules

companynative dna

companythe solutioncopyright

planar basescopyright