DNA fingerprinting

– DNA sequence “alleles” – show variation among

– related species – used to establish phylogeny

– species – used to study hybridization

– populations – used to study migration

– individuals – paternity/maternity

– breeding success

– lawsuits

– forensic applications

Major techniques:

restriction fragment length polymorphisms (RFLPs)

– differences in the size or number of restriction fragments

– caused by – point mutations of restriction sites

– create/destroy sites

– insertions/duplications

– change distance between sites

– deletions – change distances or delete sites

– inversions/translocations – change linkage of sites

– assayed using:

– restriction digests

– Southern blots with specific labelled probes

– advantages – easy to do (once probes identified)

– disadvantages – slow (Southern results)

– expensive probes

– few alleles for most sites

multilocus minisatellite DNA fingerprinting

– use minisatellite DNA

– multiple copies of short tandem repeat elements

– repeat elements < 65 bp

– caused by – different numbers of sites containing repeats

– different numbers of repeats within site

– hot-spots for recombination

– assayed using – restriction digests

– Southern blots with specific labelled probes

(for minisatellite repeats)

– identify all DNA fragments containing minisatellites

– advantages – highly polymorphic alleles

– easy to do (once probes identified)

– disadvantages – slow (Southern results)

– expensive probes

– different sites may be on same-sized DNA fragments

– confusing results

– high mutation rate (10^–2) – problem for paternity tests

microsatellite markers

– use microsatellite DNA

– multiple copies of very short tandem repeat elements

– repeat elements 1-6 bp (usually 2-4)

– e.g. (CA)_n, (AAT) _n

– caused by – DNA polymerase slippage during replication

– assayed using

– PCR primers flanking specific microsatellite sites

– primers identified by cloning and sequencing sites

– then use primers to amplify different-sized alleles

– for specific site

– compare size of PCR products – polyacrylamide gel

– use sequence product ladder as size standard

– can get size of bands to exact base length

– advantages – highly polymorphic alleles

– see all alleles for site (codominant)

– fast & easy to do (PCR)

– lower mutation rate than minisatellites

– disadvantages – need to find good PCR primers

random amplified polymorphic DNA (RAPD)

– do PCR using single random 10 base primer

– if primer hybridizes to 2 sites (opposite direction)

– and sites close enough for PCR – get PCR product

– get small number of PCR products

– variable number & size

– caused by – mutations in primer annealing sites

– DNA rearrangements (like RFLP)

– advantages – PCR – lots of product

– visible on gel without probe

– disadvantages – hard to standardize

– results vary depending on reaction conditions

amplified restriction fragment polymorphism (AFLP)

– combination of RFLP & PCR

– digest DNA with 2 restriction endonucleases

– ligate to 2 different adapters – 1 for each enzyme

– PCR using 2 different primers

– match adapter + RE site + 2 more specific bases

– so amplified products must have 2 different RE ends

– and specific DNA sequence next to RE site

– only small number of fragments get amplified

– but get lots of product for those

– easy to separate and see bands on gel

– advantages – PCR – lots of product

– visible on gel without probe

– more polymorphic than standard RFLP

– looking at more RE sites

– disadvantages – ?

DNA/amino acid sequence variation

– e.g. “molecular clock”

– major phylogenetic tool

– mitochondrial genome (maternal inheritance)

– families of related genes

– study rates of evolution