1. Simulating Genetic Screening
Gel Electrophoresis
Simulating Genetic Screening

2. Polymerase Chain Reaction (PCR)
PCR is a method for making many copies of a targeted DNA sequence in a test tube
PCR quickly copies and amplifies a few strands of DNA so the DNA can be manipulated and analyzed
There are many similarities between PCR and DNA replication, in summary:
Denaturation Step: 2 strands of DNA are separated
Annealing Step: primers “anneal” or “base-pair” in preparation for DNA replication
Extension Step: DNA replication occurs

3. Genetic Engineering: analysis of DNA fragments
Gene Splicing: DNA from one organism can be combined with the DNA of a different organism
DNA Sequencing: determine precise order of nucleotides in a DNA molecule
Gene Localization: determine exactly where genes are located within a genome
Forensic DNA Matching: determine the identity of a deceased person
DNA Fingerprinting: aid in the solving of crimes

4. Cutting DNA
DNA is very large and must be cut into smaller pieces to be analyzed
Restriction Enzymes – made from proteins and come from bacteria. They are used to cut larger molecules of DNA
1,000s of restriction enzymes and they are named for the bacteria they come from
Each restriction enzyme recognizes a specific nucleotide sequence in DNA, and cuts the DNA at the “restriction site”

5. Restriction Enzymes for Lab
EcoRI (E. coli)
HindIII (H. influenzae)
Lambda DNA: bacteriophage (bacterial virus) that infects the 2 bacteria (Harmless to humans!)

6. Restriction Sites
Palandromic: sequences of bases reads the same forward and backward (ex)Racecar
EcoRI : GAATTC
CTTAAG
HindIII : AAGCTT
TTCGAA

7. Gel Electrophoresis
Separates and analyzes different sizes of DNA fragments
Electric voltage is applied to a gel
Negatively charged DNA moves to toward the positive end of the chamber
Smaller DNA fragments will move faster and farther (will leave a banding pattern)
Stains will make the bands of DNA visible
The order of the bands in the gel tells the exact sequence of bases in the DNA

8. DNA Marker
Lambda DNA cut with the HindIII and EcoRI restriction enzymes will be our DNA marker
The genome from the bacteriophage lambda, a virus that infects E. coli
When cut with the restriction enzymes, the lambda genome will yield DNA fragments of different sizes; from 21,226 bp to 125bp. (base pairs)
3530 bp fragment may not be visible because the ends of the fragment have overhanging strands of DNA which are capable of annealing
If annealing occurs the fragments will combine to form a single 24,756bp fragment.
The marker will be heated to 65 degrees Celcius for 10 minutes prior to loading the gels to ensure separation

9. Estimate Fragment Size
Determine the approximate sizes of each of your restriction fragments. This can be done by comparing the fragments with the DNA fragments of known sizes.
Measure the distance (in millimeters) that each fragment traveled from the well.
Estimate its size, in base pairs, by comparing its position to the Lambda DNA marker.

10. Mathematical Formulas to Determine Fragment Size (more accurate)
Formulas have been developed for describing the relationship between the molecular weight of a DNA fragment and its mobility (how far it runs in the gel)
Graph the size of the known fragments from the marker against the distance each DNA band moved through the gel to generate a “standard curve”.
X axis is distance traveled in mm Y axis is size of fragment in base pairs
Use a vertical line from X axis to the point where your unknown fragment intersects the standard curve. This will determine the sizes of your unknown fragments.

11. Lab Procedure Day 1 Guided Inquiry Lab Background Pre- Lab Questions

12. Lab Procedure Day 2 Introduction to Inquiry Lab
Write up a proposal including: testable question, hypothesis, procedure, and method of data collection.
Pouring an Agarose Gel – use one of 3 methods
Loading the Gel
Running the Gel
Staining the Gel

13. Lab Procedure Day 3 Answer Lab Questions from previous day
Read Scenario: Guided Lab – Simulated Genetic Screen
Answer Pre-Lab Questions
Perform the Guided Lab procedure

14. Lab Procedure Day 4
Calculate the Relative Mobility for each of the DNA fragments
Complete the Data Table
Plot the Standard Curve
Use the Standard Curve to calculate the sample DNA fragments from Child 1 and Child 2
Answer Lab Questions and Analysis