Forensic DNA Fingerprinting: Using Restriction Enzymes Forensic DNA Fingerprinting Kit Advantages Standards Based Aligns with AP Biology Lab 6 Use of real restriction enzymes and electrophoresis of real DNA fragments Lab can completed in two 45 minute sessions Sufficient materials for 8 student workstations
The Forensic DNA Fingerprinting Kit Can Help You Teach: DNA structure DNA restriction analysis (RFLP) Agarose gel electrophoresis Molecular weight determination Simulation of DNA Fingerprinting Plasmid mapping DNA
Fingerprinting Real World Applications Crime scene Human relatedness Paternity Animal relatedness Anthropology studies Disease-causing organisms Food identification Human remains Monitoring transplants
Workshop Time Line Restriction digest of DNA samples Introduction to DNA Fingerprinting and RFLP analysis Electrophoresis on Agarose gels Analysis and interpretation of results DNA Fingerprinting Procedure Overview Laboratory
Quick Guide DNA Fingerprinting Procedures Day One DNA Fingerprinting Procedures Day Two DNA Fingerprinting
Procedures Day Three DNA is Tightly Packaged into Chromosomes Which Reside in the Nucleus Model of DNA DNA is Comprised of Four Base Pairs
Deoxyribonucleic Acid (DNA) DNA Schematic O Phosphate O P O
O CH2 Base O Sugar O Phosphate O P
O Base O CH2 O Sugar OH What
information is in our DNA? One copy of Homo sapiens DNA contains three billion base pairs located on 23 chromosomes. This amount of DNA could code for three million genes of average size (1000 bp). However, in reality, it only codes for approximately 50,000 genes. So, only a few percent of our DNA actually codes for protein! We don't know why this is so but it does provide us with some interesting observations on the sequence
content. DNA that codes for protein. These are the exons of genes ~ 2-3% Non-coding single copy, non-repetitive~ 68% Non-coding repetitive DNA ~ 30%. Of this: 2/3 Randomly repetitive (or 20% total DNA) 1/3 Tandemly repetitive (or 10% total DNA) 1985 One type of this last group is called Variable Number Tandem Repeats or VNTR. This DNA is: Non-coding and 9100bp in length Repetitive
The repeating sequence is repeated next to itself (tandem) The number of repeats is variable from person to person The VNTR DNA can be used in one method of obtaining a DNA fingerprint. This method is called Restriction Fragment Length Polymorphism and results in an RFLP Fingerprint Other Repetitive Units ---1991 Many non-coding regions have no
known function, and are sometimes referred to as "junk DNA." Microsatellites are one example of such "junk DNA. Microsatellites, or Simple Sequence Repeats (SSRs), are polymorphic loci present in DNA that consist of repeating
units of 1-6 base pairs in length Microsatellites occur in "noncoding" DNA, that is, DNA that is not transcribed into messenger RNA to code for proteins. STRs are similar to SSRs however the number of repeating bases are less (usually
2-6 bp in length) The number of repeats of the SSRs is highly variable between individuals. 2-4 STR-PCR DNA Restriction Enzymes
Evolved by bacteria to protect against viral DNA infection Endonucleases = cleave within DNA strands Over 3,000 known enzymes Enzyme Site Recognition Restriction site Palindrome
Each enzyme digests (cuts) DNA at a specific sequence = restriction site Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC) Fragment 1 Fragment 2
5 vs 3 Prime Overhang Generates 5 prime overhang Enzyme cuts Common Restriction Enzymes EcoRI Eschericha coli
5 prime overhang Pstl Providencia stuartii 3 prime overhang The DNA Digestion Reaction Restriction Buffer provides optimal conditions NaCI provides the correct ionic strength Tris-HCI provides the proper pH Mg2+ is an enzyme co-factor
DNA Digestion Temperature Why incubate at 37C? Body temperature is optimal for these and most other enzymes What happens if the temperature is too hot or cool? Too hot = enzyme may be denatured (killed) Too cool = enzyme activity lowered, requiring longer digestion time Restriction Fragment
Length Polymorphism RFLP Allele 1 PstI EcoRI CTGCAG GAGCTC GAATTC
GTTAAC 1 Allele 2 2 3 CGGCAG GCGCTC Different
Base Pairs No restriction site GAATTC GTTAAC 3 Fragment 1+2 M Electrophoresis of restriction fragments M: Marker
A-1: Allele 1 Fragments A-2: Allele 2 Fragments + A-1 A-2 Agarose Electrophoresis Loading Electrical current
carries negativelycharged DNA through gel towards positive (red) electrode Buffer Dyes Agarose gel Power Supply Agarose Electrophoresis Running
Agarose gel sieves DNA fragments according to size Small fragments move farther than large fragments Gel running Power Supply Analysis of Stained Gel Determine
restriction fragment sizes Create standard curve using DNA marker Measure distance traveled by restriction fragments Determine size of DNA fragments Identify the related samples Molecular Weight
Determination Distance (mm) 23,000 11.0 9,400 13.0 6,500
15.0 4,400 18.0 2,300 23.0 2,000 24.0
100,000 10,000 Size, base pairs Size (bp) Fingerprinting Standard Curve: Semi-log B 1,000
100 0 5 10 15 Distance, mm 20 A
25 30 DNA Fingerprinting Lab Extensions Independent studies Plasmid DNA isolation (mini-preps) Plasmid mapping using restriction enzymes Southern blot analysis Introductory labs to electrophoresis:
Kool-Aid/FastBlast pH indicator in buffer Plasmid Map and Restriction Sites 863bp 863bp 3469bp 2027bp Laboratory
Extensions BamHI Hind III EcoRI EcoRI+ HindIII 721bp 721bp 947bp
7367bp 1659bp 2027bp 6504bp BamHI: 1 linear fragment; 7367bp EcoRI: 2 fragments; 863bp / 6504bp 3 fragments; 721bp/2027bp/3469bp HindIII: EcoRI+Hind III: 5 fragments; 721bp/863bp/947bp/1659bp/2027bp
Bio-Rads Electrophoresis Equipment PowerPac Mini PowerPac Basic Electrophoresis Cells Power Supplies Precast Agarose Gels PowerPac HC
Mini-Sub Cell GT PowerPac Universal Wide Mini-Sub Cell GT
