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