1. 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

2. 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

3. Link to electrophoresis

4. 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.

5. Note that mutant lacks one of the restriction cut sites
Fig
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

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

7. 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

8. 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

9. 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

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

11. 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

12. 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.

13. 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

14. 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)

15. Figure 20.17 DNA fingerprints from a murder case

16. Link to Paternity test animation