Lab 6: D1S80 DNA Fingerprinting

Lab Objectives

• To understand how the D1S80 allele can be used to distinguish between individual human beings through the process of DNA fingerprinting

• To understand how polymerase chain reaction works (PCR)

• To learn how gel electrophoresis is used to separate DNA

Notebook

D1S80

• VNTR (variable number tandem repeat) – non coding region

• Located on chromosome 1

• Number of repeats varies from person to person (14 to 41 copies)

• Repeat is 16 bp: GAGGACCACCAGGAAG

• Length of the D1S80 region can therefore be from 224 bp to greater than 800 bp

Human Identity Techniques

• Fingerprints

• Biometry (facial measurements)

• Iris and retina patterns

• Voice recognition

• DNA fingerprinting

PCR (Polymerase Chain Reaction)

• Goal of PCR – Locate, isolate and make (“amplify”) millions of copies of a gene from one sample (equivalent to DNA replication)

• Use primers to target the gene you want to amplify

• Primers are short pieces of DNA that have a sequence complementary to either end of the target gene

dNTPS

• Deoxyribose nucleoside triphosphates

Primers

Taq polymerase

• Bacterial enzyme that is able to replicate DNA at high temperatures

• Human DNA polymerase would denature at the temperatures required by PCR

• Isolated from Thermus aquaticus in 1976 – found in hot springs and hydrothermal vents

PCR

PCR (30 cycles)

PCR Animation

Applications of PCR

• Cloning: make millions of copies of one gene from an entire genome

• DNA sequencing

• Mutagenesis: make a desired mutation in a gene of interest

• Mutation analysis: examine mutations in your genes

• Pathology: presence of harmful bacteria/viruses in your body

• Forensics: who done it?

PCR of the D1S80 Region

• Heterozygous for D1S80 – two different sized D1S80 regions

• How many bands will appear on a gel?

PCR of the D1S80 Region

• Homozygous for D1S80 – two copies of the same sized D1S80 regions

• How many bands will appear on a gel?

Procedure Overview

• Part A: Isolation of DNA from Cheek Cells

• Part B: PCR of the D1S80 VNTR region

• Part C: Electrophoresis of PCR Product

• Part D: DNA Fingerprinting Analysis

Notebook

PCR Tube Identifier _______

• D1S80 gene specific primers

• dNTPs (A, T, C, G)

• Salts (Tris-HCl, KCl, NH4Cl)

• pH of 8.9

• OneTaq® DNA Polymerase

Notebook

PCR Master Mix Components

Gel Electrophoresis of PCR Product

1. Add 2 µL of PCR loading dye to your DNA sample

2. Load 20 µL of your DNA sample into one well of a 2% agarose gel (made with Gel Green)

3. Load 20 µL of the DNA ladder into lane 5 of the gel

4. Run the gel at 70 volts for 30 minutes

Notebook

Data Table 1: DNA Marker Size

Notebook

Data Table 2: D1S80 PCR Samples

Notebook

D1S80 VNTR Analysis

• How many repeats of the D1S80 VNTR do you have on chromosome 1?

(If you are heterozygous, calculate the number of repeats for each allele)

Notebook