DNA Fingerprinting Methods

• • • • RFLP (Restriction Fragment Length Polymorphism) RAPD (Random Amplified Polymorphic DNA) PFGE (Pulse Field Gel Electrophoresis) AFLP (Amplified Fragment Length Polymorphism)

RFLP (Restriction Fragment Length Polymorphism)

Can be used for species or population identification
• Human mt DNA has 2 EcoR1 restriction sites • Honey bee mt DNA has 5 restriction sites
Q: How many bands would you see on a gel after digesting human and honey bee mtDNA with the EcoR1 restriction enzyme? Hint: mtDNA is circular in both humans and honey bees.

Can be used for analysis of relatedness

“Ladder”

Using RFLP polymorphism to study population genetic structure and evolution
Advantages: variants are co-dominant; measures variation at the level of DNA sequence, not protein sequence. Disadvantages: labor intensive; requires relatively large amounts of DNA

PCR based methods: don’t need much DNA
• RAPD: randomly amplified polymorphic DNA • AFLP: amplified fragment length polymorphism • VNTR: variable number tandem repeats; including microsatellites

PCR: polymerase chain reaction

5’ 3’

3’ 5’

RAPD: randomly amplified polymorphic DNA

Size sorted

RAPDs
Advantages: fast, relatively inexpensive, highly variable. Disadvantages: markers are dominant. Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which. Data analysis more complicated.

RAPD Analysis
B

Questions: 1. Is the locus represented by band “B” polymorphic? Band A? 2. Is individual 232 a homozygote or heterozygote for alleles represented by band “B”? What about individual 236? 3. Does band “B” represent a longer or shorter DNA fragment than band “A”.

AFLP: amplified fragment length polymorphism
Digestion of DNA with two enzymes Ligation of adapters to fragment ends Primers complementary to adapters and to 3’ region of some of the fragments

Sticky ends

AFLPs

AFLPs
Advantages: fast, relatively inexpensive, highly variable. Disadvantages: markers are dominant. Presence of a band could mean the individual is either heterozygous or homozygous for the sequence--can’t tell which.

RAPDs and AFLPs
Good for distinguishing between populations Often used for trait mapping studies because they are variable between the populations that are crossed

VNTR: variable number tandem repeats
• Non-coding regions • Several to many copies of the same sequence • Large amount of variation among individuals in the number of copies

Microsatellites
• • • • • • Not a tiny orbiting space craft Most useful VNTRs 2, 3, or 4 base-pair repeats A few to 100 tandem copies Highly variable Many different microsatellite loci (1000s) in any species

Microsatellites
• Design primers to flanking regions

Microsatellite Gels

Microsatellites
Advantages: highly variable, fast evolving, co-domininant Relatively expensive and time consuming to develop

Microsatellites
Used for withinpopulation studies; not as much for between-population studies b/c they evolve too fast Paternity analysis and other studies of kinship

Microsatellites
Questions: 1. Is the locus represented by the bands at the arrow polymorphic? 2. If it is polymorphic, how many individuals are heterozygous? 3. How many individuals are homozygous for the “short” allele?

Sequencing

Sequencing
Often used for phylogenetics (especially sequences of mitochondrial genes). Also used for studies of molecular evolution (e.g., compare rates of synonymous vs. nonsynonymous substitution)

Sequencing

Q: What’s the DNA sequence?

Amplified Fragment Length Polymorphism (AFLP)
Power + speed Sensitive at the infra-sub-specific level Highly reproducible

Genomic DNA

Restriction fragments Ligation of adapters

Digestion with Restriction enzymes

Ligated fragments DNA Fingerprint Gel analysis PCR

Amplified fragments

1

Neighbor-Joining Tree

2 3

4 5 6 7

Amplified Fragment Length Polymorphism (AFLP)
Advantages High discrimination equipment Works with all strains Automated Disadvantages Expensive Technically demanding

T-RFLP

(terminal restriction fragment polymorphism)
2) Digest labeled PCR products 3) Detect labeled terminal fragments

1) PCR-amplify and label target gene(s):

+ Restriction enzyme

Fluorescence
Fragment length (bases) Capillary electrophoresis

PCR = polymerase chain reaction

Terminal Restriction Fragment Length Polymorphism (T-RFLP)
Advantages Does not require culturing No library needed Disadvantages Expensive equipment needed Technically demanding

Pulse Field Gel Electrophoresis
Electric Field 1 + Switch Time Electric Field 2 + + + + + +

+

Pulse Field Electrophoresis (PFGE)
Advantages Used in genotyping and epidemiology High discrimination Reproducible Conclusive results Disadvantages Long assay time Limited Simultaneous strains processing

Ribotyping
# 1 #2
#3 Three E. coli isolates from environment

Isolate DNA

Cut DNA

Run DNA on gel

Probe membrane with labeled DNA to give

Ribotyping
Advantage s • Easy to type • Highly reproducible • Easy to perform • Easy to interpret • Easy to automate Disadvantag es

• Tedious • Time-consuming • Expensive