4 dna fingerprinting
TRANSCRIPT
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
1
2
3
4
5
6
7
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
ores
cenc
e
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
+
+
++ +
++
+
--
-
- --
-
-
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
##11#2#2
#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