Simulating Genetic Screening Gel Electrophoresis Simulating Genetic Screening

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

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

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”

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

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

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

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

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.

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.

Lab Procedure Day 1 Guided Inquiry Lab Background Pre- Lab Questions https://www.cff.org/

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

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

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