Advanced Techniques
Gel Electrophoresis, RFLPs and PCR. 

Human Genome Project
Gel Electrophoresis
•	Separation of DNA fragments by size
•	DNA is negatively charged
•	moves toward + charge in electrical field
•	agarose gel 
•	“swimming through Jello”
•	smaller fragments move faster
Measuring fragment size
•	compare bands to a known “standard”
•	usually lambda phage virus cut with HindIII
•	nice range of sizes with a distinct pattern
RFLP
•	Restriction Fragment Length Polymorphism
•	Due to differences in DNA between individuals
Polymorphisms in populations
•	Differences between individuals at the DNA level
RFLP use in forensics
•	1st case successfully using DNA evidence
•	1987 rape case convicting Tommie Lee Andrews
Polymerase Chain Reaction (PCR)
•	What if you have too little DNA to work with?
•	PCR is a method for making many copies of a specific segment of DNA
•	~only need 1 cell of DNA to start
PCR process
•	It’s copying DNA in a test tube!
•	What do you need?
•	template strand
•	DNA polymerase enzyme
•	nucleotides
•	primer 
PCR process
•	What do you need to do?
•	in tube: DNA, enzyme, primer, nucleotides 
•	heat (90°C) DNA to separate strands (denature)
•	cool to hybridize (anneal) & build DNA (extension)
The polymerase problem
•	Heat DNA to denature it
•	90°C destroys DNA polymerase
•	have to add new enzyme every cycle
•	almost impractical!
•	Need enzyme that can 
withstand 90°C…
•	Taq polymerase
•	from hot springs bacteria
•	Thermus aquaticus
Kary Mullis
•	development of PCR technique
•	a copying machine for DNA
Human Genome Project
•	U.S government project
•	begun in 1990
•	estimated to be a 15 year project
•	DOE & NIH
•	initiated by Jim Watson
•	led by Francis Collins
•	goal was to sequence entire human genome
•	ALL 3 billion base pairs
•	Celera Genomics
•	Craig Venter challenged gov’t 
•	would do it faster, cheaper
•	private company
Human Genome Project
On June 26, 2001, HGP published the “working draft” of the DNA sequence of the human genome.

Historic Event! 
•	blueprint 
of a human
•	the potential to 
change science & 
medicine
Maps of human genes…
•	Where the genes are…
•	mapping genes & their mutant alleles
And we didn’t stop there…
How does the human genome stack up?