Gel Electrophoresis Technique for separating DNA molecules based on size Load DNA mixture into gel containing pores of varying sizes Subject DNA to electric field; Negatively charged DNA is pulled towards positive electrode Smaller fragments travel faster than larger fragments

Smaller fragments move faster through gel than larger fragments Fig. 20-9 TECHNIQUE Mixture of DNA mol- ecules of different sizes Power source – Cathode Anode + Gel 1 Power source Smaller fragments move faster through gel than larger fragments – + Longer molecules Negative DNA moves towards the + electrode 2 Shorter molecules RESULTS Bands of DNA are visualized by the use of dyes such as ethidium bromide that bind to DNA and fluoresce pink under UV light

Link to electrophoresis

Link to RFLP RFLP – Restriction Fragment Length Polymorphism : Different lengths of DNA are produced by digesting DNA from different sources with the same restriction enzymes do to differences in number and location of restriction cut sites.

Note that mutant lacks one of the restriction cut sites Fig. 20-10 Note that mutant lacks one of the restriction cut sites Normal -globin allele Normal allele Sickle-cell allele 175 bp 201 bp Large fragment DdeI DdeI DdeI DdeI Large fragment Sickle-cell mutant -globin allele 376 bp 201 bp 175 bp 376 bp Large fragment DdeI DdeI DdeI (a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene (b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

p. 62, Problem #5 - Generating a Restriction Map Use the data given to construct a restriction map

Polymerase Chain Reaction Technique to amplify (make many copies of) short DNA sequences Based upon our knowledge of Replication Utilizes a short DNA primer complementary to beginning of sequence to be copied Utilizes series of heat and cooling steps to denature DNA strands and allow annealing of primer Utilizes heat resistant DNA polymerase

PCR vs. Gene Cloning PCR GENE CLONING Best method for increasing DNA sample when source contains minute quantities or is impure Advantage – very fast (billions of copies in hours) Disadvantage – errors accumulate as scale increases Best method for preparing large quantity of gene when purified DNA is present in sufficient quantity Disadvantage – time consuming (days) and requires pure source Advantage – DNA remains pure even as scale increases

molecules; 2 molecules (in white boxes) match target sequence Fig. 20-8 TECHNIQUE 5 3 Polymerase Chain Rxn Target sequence Genomic DNA 3 5 ( PCR) PCR is based upon our knowledge of how DNA Replication occurs. 1 Denaturation 5 3 Required: DNA to be copied (target) Primer DNA polymerase + supply of ATP, TTP, CTP, GTP 3 5 2 Annealing Cycle 1 yields 2 molecules Primers 3 Extension New nucleo- tides Use of heat –resistant DNA polymerase allows heating and cooling cycles to be automated Cycle 2 yields 4 molecules Cycle 3 yields 8 molecules; 2 molecules (in white boxes) match target sequence

Link to PCR Animation Link to summanas PCR animation Link to DNA Replication animation Link to PCR sim Utah

DNA FINGERPRINTING Technique for identifying individuals based on differences in DNA Use PCR to copy segments of DNA regions of interest Run on gel; different individuals will have different length fragments based on positions of restriction sites in their DNA

Key to easily distinguishing the DNA of different people is the noncoding repeat segments of our DNA, not mutations in genes The vast majority of our coding DNA sequences are identical in both size and sequence; generating fragments of DNA this region tends to produce fragments of identical or nearly identical size Most obvious difference in the DNA between individuals is the number of repeats in noncoding regions. Example of a repeat: [CCCATT][CCCATT] (2 copies of repeat sequence CCCATT) Individual A = 8 repeats of a segment Individual B = 4 repeats of the same segment Chances of two different people (who are not identical twins) having all same number of repeat patterns over entire noncoding region is essentially zero.

CURRENT TECHNIQUE AMPFILY REGION OF INTEREST USING PCR Case #1: 8 repeats Case #2: 4 repeats = DNA repeats = PCR primer sequences = nonrepeating DNA LOAD PCR COPIED FRAGMENTS ON GEL 100 kb 50 kb

Link to DNA interactive fingerprint VTR (Variable Tandem Repeats) – repeats of 9 to 80 letters Example two sets of 9 letter repeats: (AAATAACCGG) (AAATAACCGG) STR (Short Tandem Repeats) – repeats of 2 to 8 letter repeats: Example: four sets of 4 letter repeats: (CCCG)(CCCG)(CCCG)(CCCG)

Figure 20.17 DNA fingerprints from a murder case

Link to Paternity test animation