Gel Electrophoresis Technique for separating DNA molecules based on size

Electrophoresis- technique for separating DNA molecules based on size Fig. 20-9 TECHNIQUE Electrophoresis- technique for separating DNA molecules based on size Mixture of DNA mol- ecules of different sizes Power source Link to electrophoresis – 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

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

Link to PCR Animation Polymerase Chain Reaction Link to summanas PCR animation Link to DNA Replication animation Link to PCR sim Utah Link to PCR Animation

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

DNA FINGERPRINTING Technique for identifying individuals based on differences in DNA Use PCR to copy segments of DNA regions of interest

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)

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.

Figure 20.17 DNA fingerprints from a murder case

Link to Paternity test animation