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Review questions 1. What is the difference between identification and individualization?Identification is when an object is put in a class or a group. Individualization is when that objects has certain unique properties that makes it different from all others like it. 2. What type of repetitive DNA is tested in RFLP? VNTRs 3. Why is evidence exam, the first step of a lab’s investigation, often considered by forensic scientists to be the most important part of testing?This is often considered to be the most important part of testing because any mistakes or omissions that are made during this stage can be very difficult to overcome later on, no matter how well downstream testing is performed. 4. What two laws of inheritance offer some explanation for the genetic variation that we see from person to person, even among related individuals?Mendel’s Law of Segregation and Mendel’s Law of Independent Assortment 5. Explain in your own words what 5’ to 3’ means.5’ to 3’ is a way to refer to the direction of DNA. 5’ and 3’ come from numbering the carbons in the sugar of the DNA, which is part of DNA’s building blocks. The 5’ carbon on the sugar is attached to a phosphate group, while the 3’ carbon is attached to a hydroxyl group. 6. What was one of the first types of forensic testing that attempted (but did not achieve) individualization?The ABO blood group system (discovered by Landsteiner)

7. What were some disadvantages of RFLP?RFLP is time consuming, it uses hazardous chemicals, it requires high molecular weight fragments (20,000 to 25,000 bp), it requires at least 25ng of undegraded DNA, and it is vulnerable to DNA degradation. 8. Did the DQα kit test for sequence or length polymorphisms? Using this method, would a scientist spot amplified DNA or unamplified DNA on the test strip?The DQα kit tested for sequence differences and a scientist would spot amplified DNA on the test strip using this method. 9. What are some benefits of testing STRs for forensic purposes?STRs require low molecular weight fragments (100 to 400 bp), it can be amplified by PCR, it is faster than RFLP, it uses smaller repeats which are less vulnerable to degradation , and it is more amenable to automated computerized analyses.10. Describe Locard’s Exchange Theory in your own words.Locard’s exchange Theory describes that when two people, objects, etc are in contact in some way, there will be some transfer material, whether is it skin, hair, fibers, etc. Essentially, everything will leave their mark wherever it goes, or whatever it is in contact with. 11. Describe why it still makes sense to use presumptive tests in a crime lab’s workflow. In other words, presumptive tests are a preliminary or screening test. This type of test asks the question “could this be the target substance?”. These tests usually have a high sensitivity, but not as good specificity. 12. At what point during OCME’s testing is the presence of human blood confirmed? Human blood is based on a positive screening test for blood followed by the detection of human DNA.

13. Name one reason why semen fluoresces under an alternate light source.Sperm which fluoresce and/or Pseudomonas florescens w hich then fluoresce in the visible light spectrum. 14. Why would it make sense to continue DNA testing on a suspected semen stain that tests negative for AP?The Acid Phosphatase can lose activity after 3 months. 15. Describe how the different parts of a sperm are stained using the Christmas Tree stain. The sperm head will be stained red and the sperm tails will be stained green. 16. In what type of extraction are samples capable of being added directly to the PCR reaction?In an FTA extraction samples can be directly added to the PCR reaction. 17. Name some advantages and disadvantages of the organic extraction.Advantages of organic extraction are that it yields high quality, high molecular, and clean DNA which is necessary for RFLP. Disadvantages are that PCIA needs to be used in a chemical hood, it is time consuming and it requires multiple transfers. 18. Describe the science that explains how the Chelex extraction works.In this type of extraction, the sample are boiled to release DNA. Commercially available Chelex resin adheres to harmful polyvalent metal ions. The nucleases are then inactivated. Incubation at 56 degrees will lyse cell membranes and incubation at 100 degrees will destroy proteins and denature DNA. A final centrifugation step will pull beads and debris to botton and the ssDNA will be left on top. 19. What special reagent is added to a

differential extraction in order to lyse sperm heads? DDT breaks down protein disulfide bonds which are present in sperm heads. 20. Describe why pH is an important factor in solid phase extractions.pH is an important factor in solid phase extractions because optimized buffers lyse cells before the DNA binds the silica-coated particle in a high salt environment. Low pH favors adsorption and high pH favors elution. 21. Why is quantitation an important part of DNA testing?Quantitation is important because high levels of DNA create interpretation challenges because there are more artifacts to review. High levels of DNA quantity will also take more cycles to copy. Stochastic effects when amplifying low levels of DNA produces allele dropout. Low levels of DNA quantity will take fewer cycles to copy. 22. What was the main disadvantage of the slot blot assay?Slot blot is time consuming, laborious, subjective, ambiguous, and poor LLD (7.5 pg/ul) 23. What is different about qPCR compared to end-point PCR?qPCR allows quantification as template is doubling, which is the exponential phase and end-point PCR is based on the plateau phase of amplification. 24. Name and describe the three phases (not cycles) of a PCR reaction. Exponential: 2n (geometric increase of DNA)Linear: Arithmetic increase of DNAPlateau: reaction coming to a halt 25. Name some flaws of earlier quantitation methods that are overcome by qPCR.With PCR, after only about 30 cycles, one template can become a billion new

copies, while other earlier quantitation was time consuming. Multiplex PCR even allows several templates to be copied simultaneously. Minute amounts of DNA template are sufficient with PCR. 26. Why does a qPCR probe not fluoresce when it is intact?Energy transfers from reporter to quencher only when the probe is intact. Quencher dye inhibits fluorescence by FRET. 27. Name some reasons why a PCR reaction reaches plateau phase.The reaction can stop and reach the plateau phase because of reagent exhaustion, excess product, reannealing of product strands, competition for reactants by non-specific products, and incomplete denaturation of template. 28. Explain why it makes sense for PCR reactions to be carried out with such small volumes.With smaller volumes in a smaller container, there will be more room for the DNA to be copied. 29. What are some factors that must be considered when designing PCR primers? The length must be 18-30 bases,...