DNA+FingerPrinting



Help us solve this tragedy. Download the Pre-lab assignment. Follow the steps on this page, complete the readings and activities, and then hand in your answers via edmodo. toc
 * Who took out Mr V? **
 * media type="custom" key="8482090" || media type="custom" key="8508234" ||

= Step 1 - A little History=

DNA fingerprints may be used to help determine guilt or innocence, paternity, the evolutionary relationships between organisms, and for isolating genes from a genome. Our focus in this activity will be a Crime Scene Investigation. Read the following article about the inventor of DNA Fingerprinting Technology.

Assignment Questions: 1) Why is Alec Jeffreys concerned about DNA fingerprinting? What is his position on creating a national DNA database?

= Step 2 - PCR =

DNA can be acquired from any source containing human cells (blood, saliva, semen, hair, skin….). The lab we are about to do is a simulation of the creation and analysis of a DNA fingerprint. Your job is to become familiar with the steps associated with making a DNA fingerprint and be able to describe the function of the tools used.


 * [[image:pcranimatie.gif width="420" height="208"]] || In most crime scenes the source of human cells is very limited (drop of blood, a few body hairs, skin under the finger nails of the victim….). In order to create a DNA fingerprint a large amount of DNA is needed. It becomes necessary to take the small amount of DNA collected and make millions of copies of it. This requires the use of a special device called a PCR machine (aka: thermal cycler). The collected DNA is placed in small reaction vessels along with all four types of DNA nucleotides and DNA polymerase. ||
 * media type="custom" key="8482360" || Watch this animation of PCR. Pay attention to the Temperature! ||
 * [[image:PCR.jpg width="444" height="311" link="@http://molo.concord.org/database/activities/260.html"]] || Click on the animation to the right. From the menu jump to Polymer Chain Reaction. Go through all 5 steps paying attention to the relationship between Temperature and molecular behavior.

Don't stress over getting it right. That would take forever. Just play with it until you get the concepts.

Hint: READ the little text boxes ||

Assignment Questions: 2- Why is the PCR machine also known as a thermal cycler? 3- Why is DNA polymerase needed to support the PCR reactions? 4- What happens to the amount of DNA in a PCR machine each time it completes one cycle? 5- What is key to building a good primer

= Step 3 - Restriction Enzymes! =


 * [[image:sticky_ends.jpg]]



 || After copying large amounts of DNA using the PCR machine, the next step is to mix one or more restriction enzymes with the DNA. Restriction enzymes (aka: restriction endonucleases) can bind to DNA molecules wherever a specific sequence of nucleotide bases exist on the DNA strand. Once locating the site where the sequence exists, the enzyme cuts through the DNA. Some restriction enzymes cut straight through and create blunt end fragments while others cut through leaving exposed bases. This type of cut is referred to as a “sticky end” cut. It is called this because another DNA fragment having the complimentary sequence of exposed bases can attach to the end of the DNA. “Sticky end” cutting enzymes are what are used when attempting to splice a DNA fragment of one organism into the genome of another.

Restriction enzymes were discovered in bacteria cells. It appears that bacteria use these enzymes as a type of immune system. Any foreign DNA that enters a bacteria cell, from a virus perhaps, if the same sequence of bases is present on the foreign DNA as can be recognized by the enzyme, then the foreign DNA will be cut into pieces and rendered harmless. Many restriction enzymes have been discovered. A few are shown in the image on the left.  Their name is derived from the species of bacteria in which the enzyme was first discovered.

Restriction enzymes are added to the copied DNA so that it may be cut at multiple places and as a consequence produce many DNA fragments of varying lengths. These are called restriction fragment length polymorphisms or simply “RFLP’s”.  Once placed in a gel of a gel electrophoresis chamber the fragments will be spaced out as a result of their varying lengths (largest move the shortest distance and smallest move the greatest distance). DNA from the same source should have the same separation pattern.

All human DNA is 99.9% identical. But 0.1% is variable. This usually occurs in non-protein coding regions (non-genes). The two major types are Variable Nucleotide Tandem Repeats (VNTRs) pictured to the left and Single Nucleotide Polymorphisms (SNPs) pictured below.

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 * The Video on the right is a preview of the cloning experiment we will do and shows how Restriction enzymes are used! || media type="custom" key="8484722" ||  ||

Assignment Questions: 6- Explain the natural role of Restriction Enzymes in bacteria? 7- Explain why "Sticky End" cutting enzymes are necessary for supporting the movement of DNA fragments into the genomes of other organisms. 8- How do restriction enzymes allow us to distinguish one persons DNA from another's DNA? 9- Look at the base sequence on the DNA molecule below. How many places would the restriction enzyme EcoR1 cut this piece of DNA? How many nucleotides long is each DNA fragment?

10- How many DNA fragments would be produced if you mixed the DNA molecule above with HindIII?

=Step 4 - The Case!=


 * <span style="color: #0549a4; font-family: 'Trebuchet MS',Helvetica,sans-serif; font-size: 130%;">In this activity you will perform electrophoresis on purified DNA samples that were collected from a crime scene at our school. Mr. Beal and Mr. Jayo found Mr. Vinogradov brutally beaten and bleeding along the back of his head. Apparently the cowardly attacker approached Mr. Vinogradov from behind as he left his room in the late afternoon. This assailant hit him with a blunt object that left impressions in the scalp of Mr. Vinogradov. The pattern of the impressions revealed what appear to be rows of tiny key pads.

<span style="color: #055706; font-family: 'Trebuchet MS',Helvetica,sans-serif; font-size: 130%;">You are interning for the Bucks County CSI team and were called to the scene of the crime. After several hours of carefully collecting evidence from the crime scene you find a calculator in the bushes near the faculty parking lot that has blood on the key pad side of the calculator and a small amount of flesh in the groove of the battery compartment on the other side.The DNA of the blood matches Mr. Vinogradov’s DNA but the DNA in the flesh sample does not. You conclude that the flesh came from the perpetrator's hand as he committed this heinous act. It is a well known fact that Tennent math teachers, Mrs. Martin and Ms. Konell, have been very jealous of the attention that the Tennent Science department continually receives, especially Mr.Vinogradov and Mr. Beal. ||

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 * [[image:CSI3.jpg width="247" height="330"]][[image:CSI4.jpg width="246" height="328"]] || <span style="color: #800080; font-family: 'Trebuchet MS',Helvetica,sans-serif; font-size: 130%;">Two months ago Mr. Vinogradov and Mr. Beal launched the iPad initiative; to replace all textbooks and peripheral devices with iPads. This would include eliminating the need for the annual purchase of expensive graphing calculators. Both Mrs. Martin and Ms Konall used to work for T.I. and it is rumored that they might be profiting from these contracts.

<span style="color: #0022ff; font-family: 'Trebuchet MS',Helvetica,sans-serif; font-size: 130%;">The morning before the attack students overheard these teachers making negative comments to the Math Coordinator Mrs. Stek about both Mr. Vinogradov and Mr. Beal. Of particular concern were the comments “Apple Fan Boy” and “It’s time to make Apple Sauce”. You believe that either one of these dastardly despicable Math teachers could be the perp. You have requested the CSI team to take tissue samples for DNA comparison to the flesh taken from the calculator. The CSI team agreed but also took samples from those who were first to arrive at the scene as well. DNA for all samples was cloned in a PCR machine and put into separate reaction vessels.

<span style="color: #055706; font-family: 'Trebuchet MS',Helvetica,sans-serif; font-size: 130%;">To keep costs down instead of cloning all the DNA collected you ordered the lab tech's to use two pairs of primers so that only small portions of the teachers DNA was cloned. You anticipate that this will lead to a gel that has clearly defined bands that allow you to match the suspect and crime scene samples. ||

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