http://bcs.whfreeman.com/lehninger5e/

DNA - Deoxyribonucleic Acid

RNA Ribonucleic Acid

Discovering the structure of DNA Structure was discovered in 1953 by James Watson and Francis Crick

Outline for Replication A. Initiation B. Priming C. Elongation D. Proofreading and Termination

55335533Watson/Crick proposed mechanism of DNA replication

1955: Arthur Kornberg

Worked with E. coli. Discovered the mechanisms of DNA synthesis.

Four components are required:

dNTPs: dATP, dTTP, dGTP, dCTP(deoxyribonucleoside 5-triphosphates)(sugar-base + 3 phosphates)

2.DNA template

DNA polymerase (Kornberg enzyme)4.Mg 2+ (optimizes DNA polymerase activity)

1959: Arthur Kornberg (Stanford University) & Severo Ochoa (NYU)

SequencingSequencing is the process by which you determine the exact order of the nucleotides in a given region of DNA.Dideoxynucleotide sequencing is done through complementary chain synthesis and early termination.The synthesized chains are visualized by methods using:Radioactive labels.Nonradioactive labels.

Requirements for Sanger-Coulson SequencingDNA to be sequenced must be in single strand form.The region to be sequenced must be 3 flanked by known sequence.Reagents needed are:A primer complementary to the known region to direct chain synthesis.DNA polymerase.4 deoxynucleotide triphosphates (dNTPs).4 dideoxynucleotide triphosphates (ddNTPs).

DideoxynucleotidesdATPddATPThe 3 hydroxyl has been changed to a hydrogen in ddNTPs, which terminates a DNA chain because a phosphodiester bond cannot form at this 3 locationHere is an example comparing dATP and ddATP:

** Since the 3 OH is changed to a H in ddNTPs, it is unable to form a phosphodiester bond by nucleophilic attack on the phosphate, and it will cause a termination in the DNA chainMechanism of DNA polymerization

Sequencing Visualization MethodsTwo forms of labeling:Radioactive Primer labeled (32P or 33P)dNTP labeled (35S)Nonradioactive Primer labeledddNTP labeled (big dye terminator)

Radioactive Primer Labeled SequencingRemember each reaction has many molecules each one incorporating its respective ddNTP and stopping at a different length.

Gel SeparationThe reaction mixtures are separated on a denaturing polyacrylamide gel.Denaturing to prevent the DNA from folding up on itself while it travels through.Polyacrylamide to separate the strands which differ in length by only one nucleotide.Each band corresponds to a sequence of DNA which was terminated by a particular ddNTP.This ddNTP is identified by lane in the radioactive method and by color in the fluorescent method.The lowest band on the gel is the shortest. The shorter the strand, the earlier in the synthetic reaction the ddNTP was incorporated.The lowest band on the gel is at the 5 end of our synthesized strand and is complementary to the 3 end of our unknown fragment.

Gel VisualizationRadioactive method which requires four gel lanes, one for each reaction vessel.Readout is done by hand or with a densitometric scanner.Nonradioactive fluorescence sequencing requires only one gel lane because each nucleotide has a distinct color.The readout process is done by laser scanner and recorded by computer.

vs.Gel Electrophoresis and Readout of Reaction Products:

Polymerase Chain Reaction (PCR) and Its Applications

What is PCR?It was invented in 1983 by Dr. Kary Mullis, for which he received the Nobel Prize in Chemistry in 1993.PCR is an exponentially progressing synthesis of the defined target DNA sequences in vitro.

What is PCR? : Why Polymerase?It is called polymerase because the only enzyme used in this reaction is DNA polymerase.

What is PCR? : Why Chain?It is called chain because the products of the first reaction become substrates of the following one, and so on.

What is PCR? : The Reaction Components1) Target DNA - contains the sequence to be amplified. 2) Pair of Primers - oligonucleotides that define the sequence to be amplified.

3) dNTPs - deoxynucleotidetriphosphates: DNA building blocks.4) Thermostable DNA Polymerase - enzyme that catalyzes the reaction5) Mg++ ions - cofactor of the enzyme6) Buffer solution maintains pH and ionic strength of the reaction solution suitable for the activity of the enzyme

The ReactionTHERMOCYCLERPCR tube

Denature (heat to 95oC)Lower temperature to 56oC Anneal with primersIncrease temperature to 72oC DNA polymerase + dNTPs