1. Used for detection of genetic diseases, forensics, paternity, evolutionary links Based on the characteristics of mammalian DNA Eukaryotic genome 1000x larger than bacterial genome DNA divided into 3 classes Nonrepetitive DNA Moderately repetitive DNA Highly repetitive DNA- called SATELLITE DNA DNA Fingerprinting

2. 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

3. Applications of blot hybridization DNA Fingerprinting

4. 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 proteins Hypervariable 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 2 use semen or blood found at a crime scene as the source of DNA DNA Fingerprinting

5. 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 classes Nonrepetitive DNA - 50% of DNA, occurs in only 1-20 copies per haploid, contains almost all the genes that code for proteins Moderately repetitive DNA - contains sequences represented 20-10000x, contains a few genes including those that code for ribosomal RNA, has long stretches of nongenic DNA Highly repetitive DNA - contains units that are found 10 5 -10 7 x per haploid, no genes, called SATELLITE DNA SATELLITE DNA found in almost all eukaryotes (low amounts in humans/chicken - high amounts in kangaroo rat), not in bacteria function unknown?? most are not transcribed into RNA usually found in specific spots of chromosome - centromeres and telomeres DNA Fingerprinting

6. SATELLITE DNA satellites 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 minisatellites moderately repetitive, tandemly repeated arrays (9-100 bp) organized into mean array lengths of 500-30000 bp in euchromatin microsatellites moderately repetitive, arrays of short (2-6 bp) repeats human genome contains >30,000 microsatellite loci in euchromatin mean 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

7. 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

8. 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

9. DNA Fingerprinting THE LAB: Today - digest unknown DNA sample with EcoRI and BamHI, separate on agarose gel, estimate lengths of bands Today - Eliminate the non repeating DNA sequences Start today, finish next lab period - Ethanol precipitate Next lab period - Separate remaining DNA on agarose gel

10. 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 4 After heating, allow DNA to slow cool highly repeated DNA (satellite DNA) renature rapidly within a short 20 minutes non 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

11. USEFUL WEBSITES Folding of RNA molecules http://www.bioinfo.rpi.edu/applications/mfold/old/rna/form1.cgi Molecular Biology tools http://www.molbiol.net/ http://www.molbiol.net/biolinks/alphabar/PCR%2520and%2520Primer%2520Design.shtml http://ntdb.chem.cuhk.edu.hk/tools.htm Oligonucleotide calculator http://members.aol.com/_ht_a/lucatoldo/myhomepage/JaMBW/3/1/9/index.html http://www-medlib.med.utah.edu/masspec/mongo.htm http://www.schepartzlab.yale.edu/ Protein information http://www.basic.nwu.edu/ Chemistry calculators http://users.pandora.be/educypedia/education/calculators.htm Good websites of “tools” http://www.csb.yale.edu/people/steitz/Toolkit/toolkit.htm http://szewczak.com/ Plasmid information MacPlasmap