Download Ways to detect unique sequences within mammalian DNA

DNA Fingerprinting
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
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 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
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 105-107x 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
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
DNA Fingerprinting
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
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 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
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
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