Sequencing and Copying DNA

Gel electrophoresis Used to separate DNA fragments by size Parts Agarose gel (sponge-like matrix) Electric current (pushes the molecules through the gel) Buffer solution (maintain the pH and provide ions to conduct the current) DNA fragments Dye (usually ethidium bromide)

How it works Charged DNA molecules move from the negative end to the positive end of the gel (down from the wells) Smaller pieces travel faster/farther than larger ones

The gel is then stained The size of the DNA fragments can be determined by comparing them to markers of known length The DNA pattern can also be used to match DNA samples

DNA Sequencing Determines the sequence of the nucleotides Dideoxynucleotides are used because they will stop the addition of nucleotides when copying DNA

The reaction mixture contains - DNA to be sequenced (template DNA) - free nucleotides - free dideoxynucleotides - an enzyme (Taq polymerase) - primers

The Process DNA is heated so the double helix denatures so it becomes single-stranded

The primers attaches to the single-stranded template DNA Taq polymerase begins adding free nucleotides to build double stranded DNA

When a dideoxynucleotide is added to the DNA by the polymerase, no further nucleotides can be added Eventually there are many pieces of DNA that end at different locations

The DNA fragments can be run through a gel and the sequence of nucleotides can be read

PCR Polymerase Chain Reaction Used to copy (amplify) DNA Thermal Cycler

PCR Ingredients DNA to be copied (template) Primers Taq polymerase dNTPs (deoxynucleoside triphosphates) Buffer solution

Stages for PCR Denaturation Annealing Elongation/Extension