1.  DNA Analysis Forensic Science 12/1/14

2. Drill  Get new packet!  From which parent does mitochondrial DNA come?  Why did Tsar Nicholas give up his throne?  How many people were missing from the mass grave?  How was that determined?  HW: Review Packet Qs #1-8

3. Objectives  IWBAT  Define DNA and explain its importance to forensic scientists  Explain how the structure of a DNA molecule informs the role and function of DNA

4. Agenda  Drill  Activity 7-4: Who are the parents?  DNA Notes  Closure

5. Activity 7-4: Who are the Parents?  To which couple does each baby belong?  How much of your DNA comes from you mother? Your father?  How much DNA do you share with a sibling?

6. DNA Notes – Add this @ top  In 1953, Francis Crick and James Watson published their paper describing the structure of DNA:  DNA is a double-helix  By separating into two strands, DNA can be replicated and information can be used  DNA codes for proteins

7. Introduction  Portions of the DNA structure are as unique to each individual as fingerprints.  The gene is the fundamental unit of heredity.  Each gene is actually composed of DNA specifically designed to carry the task of controlling the genetic traits of our cells.

8. Nucleotides  DNA is constructed as a very large molecule made by linking a series of repeating units called nucleotides.  A nucleotide is composed of a sugar, a phosphorous- containing group, and a nitrogen-containing molecule called a base.  ADD THIS: There are two types of bases—purines and pyrimidines

9. ADD: Purines & Pyrimidines  Pyrimidines: Thymine and Cytosine  Purines: Adenine and Guanine C C C C C C O N C H H O N H H3CH3C H H H H N N N H O C HH N H C N N N N C C C C H H N N H C C N C H N C N HC O H H Thymine (T) Cytosine (C) Adenine (A) Guanine (G) Purine s Pyrimidine s

10. The Bases  The bases on each strand are properly aligned in a double-helix configuration, which is two strands of DNA coiled together.  As a result, adenine pairs with thymine and guanine pairs with cytosine.  This concept is known as base pairing.  The order of the bases is what distinguishes different DNA strands.

11. DNA polynucleotide A C T G T Sugar-phosphate backbone Phosphate group Nitrogenous base Sugar A C T G T Phosphate group O O–O– O O P CH 2 H3CH3C C C C C N C N H H O O C O O H C H H H C H Nitrogenous base (A, G, C, or T) Thymine (T) Sugar (deoxyribose) DNA nucleotide Figure 10.2A DNA and RNA are polymers of nucleotides

12. DNA at Work  DNA directs the production of proteins, which are made by combining amino acids.  ADD THIS: Proteins are complex molecules that express genes, and that turns into visible traits. Example: Blue vs. Brown eyes  The sequence of amino acids in a protein chain determines the shape and function of the protein.

13. DNA at Work  Each group of three nucleotides in a DNA sequence codes for a particular amino acid.  Example: G-A-G codes for the amino acid glutamine, while C-G-T codes for alanine.  If a nucleotide is “changed,” for example a T is substituted for A and G-A-G becomes G-T-G, the “wrong” amino acid is placed in the protein (in this case: glutamine is replaced with valine).  As a result, the protein may not function correctly and this is the basis for many diseases and health issues.

14. DNA at Work Continued Met Initiator tRNA 1 2 mRNA Small ribosomal subunit Start codon Large ribosomal subunit A site U A CA U C A U G P site Figure 10.13B Start of genetic message End Figure 10.13A

15. DNA Replication  DNA duplicates itself prior to cell division.  DNA replication begins with the unwinding of the DNA strands of the double helix.  Each strand is now exposed to a collection of free nucleotides that will be used to recreate the double helix, letter by letter, using base pairing.

16. DNA Replication  Many enzymes and proteins, such as DNA polymerases, are involved in unwinding the DNA, keeping the DNA strands apart, and assembling the new DNA strands.  Polymerase chain reaction (PCR) is a technique for replicating small quantities of DNA or broken pieces of DNA found at a crime scene, outside a living cell.  The ability to multiply small bits of DNA now means that sample size is no longer a limitation in characterizing DNA recovered at a crime scene.

17. DNA REPLICATION  DNA replication  Starts with the separation of DNA strands  Then enzymes use each strand as a template  To assemble new nucleotides into complementary strands Figure 10.4A A T C G G C A T T A AT C G G C A T T A A T C G G C A T T A A T C G G C A T AT C G A C T A Parental molecule of DNA Both parental strands serve as templates Two identical daughter molecules of DNA Nucleotides

18. DNA Typing  Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats.  To a forensic scientist, these tandem repeats offer a means of distinguishing one individual from another through DNA typing.

19. DNA Typing  Tandem repeats seem to act as filler or spacers between the coding regions of DNA.  What is important to understand is that all humans have the same type of repeats, but there is tremendous variation in the number of repeats each of us have.

20. Recombinant DNA Next page in packet 1. Recombinant DNA relies on the ability of certain chemicals, known as restriction enzymes, to cut DNA into fragments that can later be incorporated into another DNA strand. 2. Restriction enzymes can be thought of as highly specialized scissors that cut a DNA molecule when it recognizes a specific sequence of bases.

21. Recombinant DNA 3. Once a portion of the DNA strand has been cut out with the aid of a restriction enzyme, the next step in the recombinant DNA process is to insert the isolated DNA segment into a foreign DNA strand, usually that of a bacterium. 4. As the bacteria multiply rapidly, copies of the altered DNA are passed on to all descendants.

22. RFLP Back to DNA Notes  Restriction fragment length polymorphisms (RFLP)are length differences associated with relatively long repeating DNA strands.  RFLP form the basis for one of the first DNA typing procedures.  Animation Animation

23. RFLP  Typically, a core sequence consists of 15 to 35 bases in length and repeats itself up to a thousand times.  The key to understanding DNA typing lies in the knowledge that numerous possibilities exist for the number of times a particular sequence of base letters can repeat itself on a DNA strand.

24. RFLP Test  Once the DNA molecules have been cut up by a restriction enzyme, the resulting fragments are sorted out by electrophoresis.  Electrophoresis: materials are forced to move across a gel-coated plate under the influence of an electrical potential.  The smaller DNA fragments will move at a faster rate on the gel plate than the larger ones.

26. RFLP Test  The fragments are then transferred to a nylon membrane in a process called Southern blotting.  To visualize the RFLPs, the nylon sheet is treated with radioactive probes containing a base sequence complementary to the RFLPs being identified (a process called hybridization).

27. A Positive RFLP Test  Next, the nylon sheet is placed against X-ray film and exposed for some time.  When the film is processed, bands appear where radioactive probes stuck to fragments on the nylon sheet.

28. RFLP Test  A typical DNA fragment pattern will show two bands (one RFLP from each chromosome).  When comparing the DNA fragment patterns of two or more specimens, one merely looks for a match between the band sets.  A high degree of discrimination can be achieved by using a number of different probes and combining their frequencies.  RFLP testing has been supplanted by PCR testing, which we’ll discuss tomorrow.

29. RFLP Test Practice  In the USA, 13 STRs (which is a newer version of RFLP analysis) are required to say that DNA matches.  So, is testing 1 or 2 RFLPs enough?  Each size of RFLP may occur in up to ¼ of the population.  RFLP Test Practice  Which Man is the Father?  The Break-In

30. Closure  Take out a WHOLE sheet of paper. Write the date on the left. You may use your notes to answer these questions; then stay in your seat until you are dismissed.  Which DNA bases are purines?  GAG codes for which amino acid?  What does PCR stand for?