Used for detection of genetic diseases, forensics, paternity, evolutionary links Based on the characteristics of mammalian DNA

Eukaryotic genome1000x larger than bacterial genomeDNA divided into 3 classes

Nonrepetitive DNA Moderately repetitive DNAHighly repetitive DNA- called SATELLITE DNA

DNA Fingerprinting

Ways to detect unique sequences within mammalian DNA

Restriction digestion of chromosomal DNA works for some organisms BUT not in mammals:EX: humans have 3 billion base pairs with 1 million restriction fragments formed from a single restriction enzyme digest - TOO difficult to isolate a single band on a gel from this large number of fragments

To characterize a specific gene use blot hybridization - see Figure 1 - WE DID THIS!!

DNA Fingerprinting

Applications of blot hybridization

DNA Fingerprinting

Applications of blot hybridization

Genome of each of us is unique (exception: identical twins)Variation in sequences between individuals is most pronounced in DNA that does not code for proteinsHypervariable regions called “polymorphic sites”

Polymorphic sites - variation due to small insertions, deletions, or point mutations in restriction sites

SO .. Restriction fragment with a polymorphic site may differ from one person to the next resulting in:

RESTRICTION FRAGMENT LENGTH POLYMORPHISMS (RFLPs)Pattern of bands on a blot hybridization sometimes called a “DNA fingerprint” because can use it to identify an individual

Uses of DNA fingerprinting - see Figure 2use semen or blood found at a crime scene as the source of DNA

DNA Fingerprinting

Ways to detect unique sequences within mammalian DNA without use of blot hybridization

Restriction digests of highly repetitive DNA form discrete bands on gel

DNA divided into 3 classesNonrepetitive DNA - 50% of DNA, occurs in only 1-20 copies per haploid, contains

almost all the genes that code for proteinsModerately repetitive DNA - contains sequences represented 20-10000x, contains a few

genes including those that code for ribosomal RNA, has long stretches of nongenic DNAHighly repetitive DNA - contains units that are found 105-107x per haploid, no genes,

called SATELLITE DNA

SATELLITE DNAfound in almost all eukaryotes (low amounts in humans/chicken - high amounts in kangaroo rat), not in bacteriafunction unknown?? most are not transcribed into RNAusually found in specific spots of chromosome - centromeres and telomeres

DNA Fingerprinting

DNA Fingerprinting

SATELLITE DNAsatellites highly repetitive with long repeat lengths (1-1000s bp)

organized as large clusters (up to 100,000,000 bp)in heterochromatin regions near centromeres and telomeres; also found on Y chromosome

minisatellitesmoderately repetitive, tandemly repeated arrays (9-100 bp)organized into mean array lengths of 500-30000 bpin euchromatin

microsatellitesmoderately repetitive, arrays of short (2-6 bp) repeatshuman genome contains >30,000 microsatellite loci in euchromatinmean array sizes of 10-100 bp

Satellite DNAs show exceptional variability among individuals, particularly with regard to # of repeats at a given loci

Minisatellite loci are most highly polymorphic and determining the repeat lengths of these loci is basis of DNA typing systems used in forensics

Ways to detect unique sequences within mammalian DNA without use of blot hybridization

restriction digestion from highly repetitive satellite DNA sequence of sheep produces 3 discrete fragments (separate by gel electrophoresis)

DNA Fingerprinting

DNA Fingerprinting

SATELLITE DNA

Sheep & cow are closely related animals so bulk of DNA is quite similar BUT

Sequences in satellite DNA are quite different from each other - see restriction digest maps Figure 3

DNA Fingerprinting

THE LAB:

Today - digest unknown DNA sample with EcoRI and BamHI, separate on agarose gel, estimate lengths of bandsToday - Eliminate the non repeating DNA sequences

Start today, finish next lab period - Ethanol precipitate

Next lab period - Separate remaining DNA on agarose gel

DNA Fingerprinting

THE LAB:

Today - digest unknown DNA sample with EcoRI and BamHI, separate on agarose gel, estimate lengths of bands***Eliminate the non repeating DNA sequences

To eliminate nonrepeating sequences heat DNA to ~100 ˚C to denature DNA - see Figure 4After heating, allow DNA to slow cool

highly repeated DNA (satellite DNA) renature rapidly within a short 20 minutesnon repetitive DNA takes hours to renature

So non repetitive DNA remains single stranded and can be digested with S1 nuclease which specifically cleaves single stranded DNA

USEFUL WEBSITES

Folding of RNA moleculeshttp://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi

Molecular Biology toolshttp://www.molbiol.net/http://www.molbiol.net/biolinks/alphabar/PCR%2520and%2520Primer%2520Design.shtmlhttp://ntdb.chem.cuhk.edu.hk/tools.htm

Oligonucleotide calculatorhttp://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.htmlhttp://www-medlib.med.utah.edu/masspec/mongo.htmhttp://www.schepartzlab.yale.edu/

Protein informationhttp://www.basic.nwu.edu/

Chemistry calculatorshttp://users.pandora.be/educypedia/education/calculators.htm

Good websites of “tools”http://www.csb.yale.edu/people/steitz/Toolkit/toolkit.htmhttp://szewczak.com/

Plasmid informationMacPlasmap