Biotech Continued….

1. Biotech Continued…. 4.4

2. How do forensic scientists determine who’s blood has been left at a crime scene? • How are paternity tests conducted? • ANSWER: Using restriction enzymes, a technique called Gel Electrophoresis, and a concept called RFLP (restriction fragment length polymorphism)

3. Individuals have differences in their DNA sequences. • Differences in coding regions (exons) may result in a mutation (ie. Sickle cell anemia) • Differences in noncoding regions (introns) may exist in the form of variations in the number of repeating units

4. RFLP analysis • If a sample of DNA is digested by a restriction enzyme, the result would be the formation of a number of DNA fragments of various lengths. • Since we all have different DNA (because we all have different numbers of VNTR) exposure to the same restriction enzyme would produce different numbers and lengths of fragments. • This is known as RESTRICTION FRAGMENTS LENGTH POLYMORPHISM

5. RFLP can be used to identify individuals. • Scenario: A crime is committed and there are 3 suspects. • If the criminal left a sample of blood at the crime scene, RFLPs from this sample can be compared to RFLPs from blood of all suspects to determine who the criminal is.

6. Gel Electrophoresis • The DNA fragments must be separated and purified from each other for analysis using a process called gel electrophoresis • In this process, the DNA fragments are placed in wells on a “jello-like gel- called agarose (or agar) • The agarose is fibrous and acts like a net • An electrical current is passed through the gel • Since DNA is negatively charged, the fragments of DNA are attracted to the positive end.

9. As the DNA fragments move through the agar, the small fragments move quickly, but larger fragments move slowly because they get caught up in the fibres of the gel. • A blue dye is added initially to the DNA fragments. When the dye reaches the positive end, the power is turned off.

10. A fluorescent dye is then used to stain the DNA fragments. • Ideally this creates a band pattern that is unique to each individual.

12. Molecular Markers are DNA fragments of known lengths. They may be run during a gel electrophoresis so that the fragments of the sample DNA can be compared to the markers to determine approximate length.

13. In reality, the amount of DNA is usually so large and the bands too numerous that instead of seeing individual bands, a large smear is seen on the gel. • In a process called SOUTHERN BLOTTING, the DNA from the gel can be transferred to a nylon membrane. • The nylon membrane is placed against an X-ray film to read an autoradiogram which will show the band pattern

15. Match the Suspect • The band pattern of suspect 1 matches the specimen. Thus, suspect 1 is probably our criminal • The specimen must also be compared to the victim because the victim’s blood may be mixed up in the specimen or it could just be the victim’s blood.

16. Who’s your Daddy??? • IN paternity test, the child’s DNA is compared to its mother’s and the possible fathers. Since the child has DNA from both its mom and dad, the bands that match its mother can be ignored. • Look at the bands that do not match the mother’s • Who does it match? • The reason the child doesn’t have the exact same DNA of its parents is because it only receives half of each parent’s chromosomes/DNA

17. PCR: Polymerase Chain Reaction • PCR is a technique used to clone (amplify) DNA • Before the technique was developed, if scientists wanted to make multiple copies of a gene fragment, the gene would have to be inserted into a plasmid and the bacterial cell would make more copies when it replicates its plasmids. Then scientists would have to remove the plasmids and cut out the bacterial genes.

18. With PCR, many copies of DNA can be made quickly. • This is particularly useful when only a small sample of original DNA is available. • (Ex: If only a small sample of DNA is obtained from a crime scene, PCR may be used to allow for multiple forensic tests.)

19. Before running a DNA through the Gel Electrophoresis for analysis, the sample will undergo PCR to make sure there is enough DNA for adequate testing. • The process of PCR is closely related to DNA replication that occurs within the nucleus of a cell.

20. DNA REPLICATION The 2 DNA strands are separated using enzymes helicase and gyrase PCR The 2 strands of DNA are separated using heat At temps of 94°C – 96 °C, the hydrogen bonds between the complimentary strand will break, separating the strands

21. DNA REPLICATION An RNA primer must be added first before DNA nucleotides are added to build a complementary strand to the template strand PCR A DNA primer is used instead because they are easy to produce in labs. (The temp is lowered to 5O°C - 65 °C to allow for the DNA primers to attach) One of the primers is known as a forward primer and the other is a reverse primer cause they start synthesis of DNA in opposite directions

22. DNA REPLICATION Once the RNA primers have been laid down, DNA polymerase III will build a complementary strand by adding DNA nucleotides PCR Temp raised to 76°C Taq polymerase – a type of DNA polymerase – builds complementary strands using DNA nucleotides that have been added to the solution @ 76°C – the Taq polymerase does not denature because it is extracted from a bacteria that lives in hot springs (Used to hot temps!)

23. When the complementary strands have been build, the cycle can repeat itself over and over again, doubling the number of copies each time.

25. Simplified process….

26. Animations • http://highered.mcgraw-hill.com/sites/0072437316/student_view0/chapter16/animations.html#