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

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.
RAPD Analysis Questions:
1. Is the locus represented by
band “B” polymorphic? Band
A?
B
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 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 + speed
Sensitive at the infra-sub-specific level
Highly reproducible
 Restriction
Genomic DNA
fragments

Digestion with Ligation of

Restriction enzymes adapters

Ligated
fragments

DNA Fingerprint
PCR

Gel analysis

Amplified
fragments
 1

Neighbor-Joining Tree 2

7
 Amplified Fragment Length
Polymorphism (AFLP)

Advantages Disadvantages
High discrimination Expensive
equipment
Works with all strains Technically demanding
Automated
 T-RFLP (terminal restriction fragment polymorphism)
1) PCR-amplify and 2) Digest labeled PCR 3) Detect labeled
label target gene(s): products terminal fragments

Fluorescence
+
Restriction enzyme
Fragment length
(bases)

Capillary electrophoresis

PCR = polymerase chain reaction

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

Does not require Expensive

culturing equipment needed
No library needed Technically
demanding
 Pulse Field Gel Electrophoresis

Switch Time
Electric Field 1 Electric Field 2

- -
- -
- -
- -
+ + +
+
+ +
+ +
 Pulse Field Electrophoresis
(PFGE)

Advantages Disadvantages
Used in genotyping Long assay time
and epidemiology
Limited Simultaneous
High discrimination strains processing
Reproducible
Conclusive results
 Ribotyping
#
1
#2

#3
Three E. coli Isolate DNA Cut DNA
isolates from
environment

Run DNA on gel Probe membrane with

labeled DNA to give
 Ribotyping

Advantage Disadvantag
s es
• Easy to type • Tedious
• Highly reproducible • Time-consuming
• Easy to perform
• Easy to interpret
• Expensive
• Easy to automate