Lab 5: Shark Attacks, Again! DNA Fingerprinting to the Rescue - - PowerPoint PPT Presentation

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Lab 5: Shark Attacks, Again! DNA Fingerprinting to the Rescue - - PowerPoint PPT Presentation

Mar 13, 2023 371 likes •660 views

Lab 5: Shark Attacks, Again! DNA Fingerprinting to the Rescue Notebook Lab Objectives Develop an understanding of the basic techniques used to study genetic polymorphisms encoded in DNA Gain familiarity with Restriction Fragment Length

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SLIDE 1

Lab 5: Shark Attacks, Again!

DNA Fingerprinting to the Rescue

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SLIDE 2

Lab Objectives

  • Develop an understanding of the basic

techniques used to study genetic polymorphisms encoded in DNA

  • Gain familiarity with Restriction Fragment Length

Polymorphisms (RFLPs) and their use in the study

  • f biodiversity
  • Apply RFLP analysis to genetic fingerprinting and

shark identification

  • Use critical thinking to solve problems through

DNA analysis

Notebook

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SLIDE 3

Reef Point, Australia

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Scenario

  • Series of shark attacks at a remote Australian

surfers’ paradise, Reef Point Beach

  • Reef Point Bay is a mating point for spotted

seals (prey for sharks)

  • Warmer waters and climate change to blame
  • Determine whether the attacks were

committed by the same shark or more than

  • ne through forensic analysis of DNA samples
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SLIDE 5

Shark Facts

  • Average yearly fatalities from shark attacks

worldwide is just 2

  • Humans kill approximately 2 million sharks per

year

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SLIDE 6

Materials

  • 2% agarose gel with Gel Green Nucleic Acid

Stain

  • 1X TBE (Tris-borate EDTA) buffer
  • 4 shark DNA samples for fingerprinting

analysis

  • 1 tube of DNA molecular weight marker (DNA

Ladder)

  • 6X gel loading dye

Notebook

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SLIDE 7

Gel Preparation

  • Prepare 50 mL of a 2% agarose gel

(In your notebook, show calculations for amount of agarose powder required and dilution of 20X TBE buffer)

Notebook

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SLIDE 8

Gel Green Dilution for Gel

  • Gel Green is 10,000x concentrated
  • 1X concentrated within the gel

(Show calculations for dilution for Gel Green in your lab notebook)

Notebook

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SLIDE 9

PreLab Questions

  • What is a DNA fingerprint?
  • What are restriction enzymes?
  • What is a restriction length polymorphism

(RFLP)?

  • How are RFLPs used as a DNA identification

method?

Notebook

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Gel Electrophoresis Applications

  • DNA fingerprinting for many purposes
  • DNA sequencing (old school method)
  • Paternity testing
  • Examine evolutionary relationships among
  • rganisms (similarities and differences)
  • Diagnose genetic disorders or gene testing
  • Determination of impurities in a sample
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DNA Fingerprinting

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VNTRs

  • Variable number of tandem repeats
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Restriction Enzymes Cut DNA

  • Cut DNA molecules at or near a specific

sequence of nucleotides

  • Creates smaller fragments of DNA
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SLIDE 14

Restriction Enzymes Cut DNA

  • Use gel electrophoresis to compare multiple

samples of DNA

  • Similar DNA samples have similar restriction

sites and will produce fragments of similar sizes

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DNA Fingerprinting

  • A DNA Fingerprint represents an individual’s

unique genetic profile

  • Analyze variable regions in DNA in order to

identify individuals

  • Scientists usually focus on regions of DNA that

are known to typically vary across individuals

  • Isolated variable regions of DNA are then

separated by size using gel electrophoresis

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DNA Fingerprint

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DNA Sequencing

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Forensics

  • Restriction enzymes and gel electrophoresis

are used to create DNA fingerprints to analyze DNA samples from crime scenes

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Paternity Testing

  • The pattern of DNA fragments

(produced by the restriction enzyme digest) is compared and if the child's DNA looks like a combination of the two parents' DNA, then the child is theirs

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SLIDE 20

Evolutionary Relationships

  • Which of the species, X or Y, is more closely

related to species Z?

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SLIDE 21

Diagnose Genetic Disease

  • Molecular diagnosis of a particular inherited

disease

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Reagents

  • DNA from Crown Beach (CB) – 15 µL
  • DNA from Doom Cove (DC) – 15 µL
  • DNA from Wave Crest (WC) – 15 µL
  • DNA from Hoppa-Hoppa (HH) – 15 µL
  • DNA Ladder (LAD) – 15 µL
  • 6X Gel Loading Dye (LD) – 20 µL

Notebook

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SLIDE 23

Procedure Overview

  • Add 3 µL of 6X Gel Loading Dye (LD) to each

DNA sample (including the DNA Ladder)

  • Mix well by gently tapping the tube on the lab

table

  • Load DNA samples onto the gel, from left to

right, as follows:

Notebook

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SLIDE 24

Results

  • Transfer the picture of your gel into your lab

notebook using the template provided

Notebook

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SLIDE 25

Ladder

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Analysis Questions

  • 1. Why is there more than one DNA band within

each sample analyzed?

  • 2. What caused the DNA to become cleaved

(broken up) in small fragments?

  • 3. Examine the shark DNA from Crown Beach.

How many restriction sites do you think existed within this sample of DNA? (assume a single linear piece of DNA was the starting material)

Notebook

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SLIDE 27

Analysis Questions

  • 4. Based on your analysis of the DNA samples,

how many sharks were involved in the recent Reef Point attacks? Explain if one or more sharks were involved, and which attacks if any may have involved the same shark.

  • 5. What chemical helps make the DNA visible

when exposed to UV light?

Notebook