Gel Electrophoresis

A molecular biology tool

Purpose

• To separate and analyze/compare fragments of DNA

4 Main Steps:

• 1) Restriction Enzymes:– Either one or several R.E.’s is added to a

sample of DNA.– Enzymes cut them into fragments

• 2) Prepare Gel:– A gel with consistency similar to gelatin (or

pGreen agar) is formed– Small comb placed halfway into one end of

gel to create DNA “wells”– Comb wiggled out leaving vacant wells

• 3) The Electric Field:– Gel placed in electric field where one end of

gel is + and one –

• 4) The Fragments Move:– DNA has negatively charged phosphates

which will move towards + end of plate– Gel provides some resistance– ** Smaller fragments move further!– (like snakes moving thru netted swimming

pool)

After gels run…

• Gel is stained with dye (ethidium bromide – carcinogenic OR methyl blue)

• Will see many bands – Bands depend on starting DNA and r.e. used

Loading Dye

• Blue colored solution used when loading DNA so you know when to shut off electric current

• Usually size marker run along with loading dye and DNA fragments

Possible analysis 2

• Can compare DNA samples run in SAME gel

DNA Fingerprints

aka DNA Profiles

Definition

• A pattern of bands made up of specific fragments from an individual’s DNA

How are they used?

• To establish whether 2 individuals are related

• To determine how closely related 2 different species are

• To help solve crimes: – Match crime scene DNA w/ suspect DNA

How to Make a DNA Fingerprint~ PCR

• Requires 20 ng DNA (can be somewhat degraded)1. Denaturization by heat2. hybridization (complementary pairing of short

primers)

http://www.accessexcellence.org/AB/GG/nucleic.html

3. DNA synthesis (by DNA polymerase)• Process repeats• http://

www.accessexcellence.org/AB/GG/polymerase.html

How to Make a DNA fingerprint

• 1) RFLP (restriction fragment length polymorphism) analysis– Involves extracting DNA from a specimen of

blood or other tissue– Cutting it into fragments using r.e.’s– Number and length of fragments vary from

person to person

What is a Polymorphism?

• Variations in DNA sequence between individuals

• sequences with the highest degree of polymorphism are very useful for DNA analysis in forensics cases and paternity testing

RFLP’s (1st generation tech.)

• Phased out in 1999 (started in ’85)• Looked at 5 areas of DNA

(non-coding/junk areas)• Required 400 ng non-degraded DNA• Time consuming, labor intensive• Took several weeks, months for results• Use radioactive labels & X ray film –

“Southern Blotting”

RFLP Allele Analysis

How to Make DNA fingerprint

• 2) Short tandem repeats – Used presently– Few base prs. (10-100) repeated a # of times– Requires 2.5 ng or less – Can use badly degraded DNA (remains)– Extremely sensitive– Automated technique ~ 2 days to a week– Highly specific profiles unique to the individual

STR’s

• Target site identified by primers binding to the correct locus

• 13 core CODIS "Short Tandem Repeat" loci used for the national DNA databank – CODIS = Combined DNA Index System

• PCR produces millions of copies of STR locus

• STR fragments separated by electrophoresis and analyzed by computer (1/2 hour)

Probability of DNA Profiles

• The probability (P) for a DNA profile is the product of the probability (P1, P2, ... Pn) for each individual locus, i.e.

• Profile Probability = (P1) (P2) ... (Pn)

• The probability can be an extremely low numbers when all 13 CODIS STR markers are included in the DNA profile!

Frequency Calculation Example

Blackett ActivityBob Blackett calculated his own profile

probability at 1.3 times 10-16, or no more frequent than 1 in 7.7 quadrillion individuals (7.7 million billion), which is more than a million times the population of the planet.

How do RFLP’s and STR’s differ?

• Restriction fragments containing STR’s vary in size among individuals b/c of differences in STR lengths, rather than b/c of diff. # of restriction sites within that region of the genome as in RFLP’s

Review

• Review electrophoresis simulation

• Forensic DNA analysis