forensic dna fingerprinting lab...forensic fingerprinting lab objectives •use restriction enzymes...
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Forensic DNA Fingerprinting Lab
Tools and Technology Used During Lab
• p20 micropipette
• Restriction enzymes (EcoRI, PstI, HindIII)
• Water bath – 37 °C
• Agarose gel electrophoresis
• DNA ladder (molecular ruler)
Restriction Enzymes(also known as restriction endonucleases)
What are restriction enzymes?
• An enzyme that cuts DNA at specific nucleotide sequences known as restriction sites
• Naturally found in bacteria and have evolved to provide a defense mechanism against invading viruses
• In bacteria, restriction enzymes selectively cut up foreign DNA in a process called restriction
Restriction Enzymes in Bacteria
Restriction Enzymes
• Cut both strands of the DNA double helix
• Over 3000 enzymes have been identified
• More than 600 available commercially
• Routinely used for DNA modification and manipulation in laboratories (“molecular scissors”)
Restriction Sites
• Also known as recognition sites
• Generally genetic palindromic sequences
• A palindromic sequence in DNA one in which the 5’ to 3’ base pair sequence is identical on both strands
• Usually a 4 or 6 base pair sequence
Restriction Sites
• The enzymes scan DNA sequences, find a very specific set of nucleotides and make a cut
Hae III
• HaeIII is a restriction enzyme that searches the DNA molecule until it finds this sequence of four nitrogen bases - GGCC
5’ TGACGGGTTCGAGGCCAG 3’
3’ ACTGCCCAAGGTCCGGTC 5’
Hae III
• Once the recognition site was found HaeIII will go to work cutting (cleaving) the DNA
Blunt Ends versus Sticky Ends• Hae III produces “blunt ends” when cleaving
DNA
• Other enzymes produce “sticky ends”
blunt end
sticky end
Restriction Enzyme Names
• Named after the type of bacteria in which the enzyme is found and the order in which the restriction enzyme was identified and isolated
EcoRI for example
R strain of E.coli bacteria
I as it is was the first E. coli restriction enzyme to be discovered.
Restriction Enzymes and Gene Cloning
Separating Restriction Fragments
• Restriction enzymes generate RFLPs (“rif-lips”) - Restriction fragment length polymorphisms
• RFLPs are differences among individuals in the lengths of DNA fragments cut by enzymes
• RFLPs can be separated using agarose gel electrophoresis and then analyzed
RFLPs(Restriction Fragment Length Polymorphism)
Separating Restriction Fragments
Gel Electrophoresis
• Gel electrophoresis is a laboratory method used to separate mixtures of DNA, RNA, or proteins according to molecular size, shape and charge
• The molecules to be separated are pushed by an electrical field through a gel that contains small pores (“molecular strainer”)
Gel Electrophoresis• Larger molecules meet with more resistance
when moving through the gel than smaller molecules
• Smaller molecules will travel farther than larger molecules
Gel Electrophoresis
More About Gel Electrophoresis
• Agarose gel – agarose is a polysaccharide extracted from seaweed
• Buffers are used to prepare and cast gels and are also used to fill the electrophoresis chamber
• The buffer acts to stabilize pH, maintain molecule shape, and conduct electricity
Nucleic Acid Stains
• DNA is not visible
• Gels are cast with a nucleic acid stain that, when exposed to UV light, will cause the DNA to fluoresce (Gel Green)
• As DNA migrates through the agarose gel, the stain will bind to the nucleotides
Forensic Fingerprinting Lab Objectives
• Use restriction enzymes to detect differences in the base sequences of individuals
• Use gel electrophoresis to analyze DNA samples from suspects and crime scene DNA
DNA Fingerprint
DNA Fingerprinting
• Technology used to analyze evidence in law enforcement cases and other applications such as:
–Paternity testing
–Determine evolutionary relationships among organisms (similarities and differences)
–Diagnose genetic disorders or gene testing
–Determination of impurities in a sample
Crime Scene DNA Analysis
Paternity Testing
Evolutionary Relationships
Procedure Overview
• DNA from crime scene and DNA from 5 potential suspects
• Part 1: Restriction Digest of DNA Samples
• Part 2: Agarose Gel Electrophoresis of DNA Samples
The Crime and Victim
Suspect 1 – Bobby Joy
Suspect 2 – Paisley Gavin
Suspect 3 – Malcolm Plum
Suspect 4 – Ella Mae Dixon
Suspect 5 – Ruby Warner
The Crime and Victim
Suspect 1 – Angelina Bento
Suspect 2 – Kay McNamara
Suspect 3 – Tori Howard
Suspect 4 – Bella Valentino
Suspect 5 – Abby Farrell
Part I: Restriction Digest of DNA Samples
• EcoRI/PstI enzyme mix (ENZ)
• Crime scene DNA (CS) - green
• Suspect 1 (S1) - blue
• Suspect 2 (S2) - orange
• Suspect 3 (S3) - violet
• Suspect 4 (S4) - pink
• Suspect 5 (S5) – yellow
• Ice and a 37 °C water bath
Part II: Agarose Gel Electrophoresis
• Add 5 uL of loading dye (LD) into each sample
• Load gel as follows:
– Lane 1 – S, DNA size standard – 10 µL
– Lane 2 – CS, green tube – 20 µL
– Lane 3 – S1, blue tube – 20 µL
– Lane 4 – S2, orange tube – 20 µL
– Lane 5 – S3, violet tube – 20 µL
– Lane 6 – S4, red tube – 20 µL
– Lane 7 – S5, yellow tube – 20 µL