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”

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

Using RFLP polymorphism to study population genetic structure and evolution

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

3’5’

5’3’

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.

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”.

B

RAPD Analysis

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 within-population 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. non-synonymous substitution)

Sequencing

Q: What’s the DNA sequence?

Amplified Fragment Length Polymorphism (AFLP)

Power + speedSensitive at the infra-sub-specific level

Highly reproducible

Genomic DNA

Digestion withRestriction enzymes

Restrictionfragments

Ligation ofadapters

Ligatedfragments

PCR

Amplifiedfragments

Gel analysis

DNA Fingerprint

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Neighbor-Joining Tree

Amplified Fragment Length Polymorphism (AFLP)

Advantages Disadvantages

High discrimination Expensive equipment

Works with all strains Technically demanding

Automated

PCR = polymerase chain reactionPCR = polymerase chain reaction

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

2) Digest labeled PCR products

+Restriction enzyme

3) Detect labeled terminal fragments

Fragment length(bases)

Flu

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Capillary electrophoresis

T-RFLP (terminal restriction fragment polymorphism)

Terminal Restriction Fragment Length Polymorphism (T-RFLP) Advantages Disadvantages

Does not require Expensive culturing equipment needed

No library needed Technically

demanding

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Electric Field 1 Electric Field 2Switch Time

Pulse Field Gel Electrophoresis

Advantages

Used in genotyping and epidemiology

High discrimination

Reproducible

Conclusive results

Disadvantages

Long assay time

Limited Simultaneous strains processing

Pulse Field Electrophoresis(PFGE)

RibotypingRibotyping

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#3#3

Three Three E. coliE. coli isolates from isolates from environmentenvironment

Isolate DNAIsolate DNA Cut DNACut DNA

Run DNA on gelRun DNA on gel Probe membrane with Probe membrane with labeled DNA to give labeled DNA to give “fingerprint”“fingerprint”

RibotypingRibotyping

• Easy to typeEasy to type• Highly reproducibleHighly reproducible• Easy to performEasy to perform• Easy to interpretEasy to interpret• Easy to automateEasy to automate

• Tedious Tedious

• Time-consumingTime-consuming

• ExpensiveExpensive

AdvantageAdvantagess

DisadvantagDisadvantageses