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Final Exam Notes

by: carla Notetaker

Final Exam Notes Bio086

Marketplace > University of Vermont > Biology > Bio086 > Final Exam Notes
carla Notetaker

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Study guide for the final exam!
into to forensic science
Dr. Yonan
Study Guide
50 ?




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This 8 page Study Guide was uploaded by carla Notetaker on Saturday September 24, 2016. The Study Guide belongs to Bio086 at University of Vermont taught by Dr. Yonan in Spring 2016. Since its upload, it has received 13 views. For similar materials see into to forensic science in Biology at University of Vermont.


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Date Created: 09/24/16
DNA: Two primary purposes: 1. Carry instructions to make proteins • Proteins carry out all functions of life • They build/are the machinery  • Different proteins à different cell types 2. Make copies of itself • So that cells can divide and replicate • Make exact replicas of all cells The Five Bases: DNA: • U = Uracil (replaces T) • A = Adenine Rules: • T = Thymine • A always binds with T • G = Guanine • with two Hydrogen Bonds • C = Cytosine • G always binds with C RNA only: • with three Hydrogen Bonds Two strands are complementary  Both strands have same information 5’­ ATG GCC GTA ATC GCA ­3’ 3’­ TAC CGG CAT TAG CGT ­5’ • Then the other strand has what sequence? Or  5’­ TGC GAT TAC GGC CAT ­3’ Because we always read 5’ to 3’ DNA Replication: Makes logical sense how DNA replication happens: 1. DNA has two strands with identical information 2. Must open up 3. Exposing unpaired bases 4. Bases are matched perfectly (compliment) 5. Forming two double helixes from one 23 Chromosome pairs 22 Autosomes Allele: Alternative form of one gene or marker; different versions Polymorphism: A silent change (something that doesn’t affect the protein), that is often common in population Mutation: A change in the DNA sequence that will change the protein’s function or regulation, usually in a  detrimental way  Homozygous – Individuals with two identical versions of a gene Same allele (yy) Heterozygous ­ Individuals with two different versions of a gene Two different alleles (Yy) Case33: Colin Pitchfork • Pitchfork was arrested and his DNA turned  • two 15 year old girls were raped and  out to be a perfect match murdered Case34: Ian Simms • First DNA fingerprinting ever done • Helen McCourt disappeared completely • Determined that the same man committed  • She was never seen again and her body has  both crimes never been found • They were saying that a man named Colin  • Gossip was that McCourt was having an  Pitchfork had paid a man he worked with to  affair with a married man named Ian Simms give a blood sample in Pitchfork’s name • Evidence included: • Therefore the police thought they had  • Scratches on Simms’ face genotyped Pitchfork already but in reality  • A scream was heard coming from his they had not pub • Bloodstained earring was found in  • Part of the civil war that led to the formation Simms’ car of the USSR • Also a strand of long hair matching  • 1991 – bodies were dug up that might be the to McCourt’s hair color Imperial Family • In his apartment there were  • First the two largest skulls were examined  bloodstains using photographs and facial reconstruction  • McCourt’s purse and coat were  – could be the Tsar and his wife  found in the village – some of it was  • DNA was taken from the remains also bloodstained • Again there was no record of the Tsar’s  • Her clothing had hair from Simms’  DNA profile dogs and fibers from Simms’  • Instead they used his living relatives apartment carpet • Prince Phillip – husband of the queen of  Case35: Dr. Joseg Mengele England • Chief medical officer from Auschwitz • The bones were matched using the  • Performed barbarous experiments on the  mitochondrial DNA profile camp’s prisoners • The royal family had been found • He evaded capture after WWII and fled to  Case37: Anna Anderson Argentina – changed his name and hid • Tsar’s daughter Anastasia's body was never  • Was reported that he died in 1979 and was  found buried in Brazil under a different name – Along with the body of her brother  • The body was exhumed from the grave Alexei • DNA was taken from the corpse and a DNA  • Idea that these two escaped has remained  profile was developed popular legend • No DNA profile was known for Mengele • Anna Anderson appeared in 1920 claiming  • Instead they took DNA from his living  to be Anastasia relatives – She knew some very specific details  • Body proved to be Mengele’s based on  about Anastasia’s life  DNA profile • DNA was taken from Anderson’s body after Case36: Tsar Nicholas II her death • July 1918 – Tsar of Russia and his  • Compared to the Tsar’s family DNA and it  immediate family were killed was proven there was no way Anderson  could have been Anastasia Isolating DNA Organic Extraction:  1. SDS – Detergent (soap) – Breaks up the fatty cell membranes 2. Proteinase K – Breaks up proteins 3. Phenol/Chloroform – Centrifuging (spinning) the tube separates the solid parts from the aqueous layer  – DNA into the aqueous layer Chelex Extraction:  • Chelating resin – Ions are absorbed into the resin – These ions can no longer degrade DNA  • DNA sample is added to resin • Boiled for several minutes – This breaks open the cells and destroys proteins and membranes • Centrifuging pulls solid parts to bottom of tube and ssDNA is left in supernatent FTA Paper Extraction:  • Absorbent paper containing: – Chemicals that protect DNA from   degradation and bacterial/mold growth • DNA absorbed onto FTA paper can be stored at room temperature for years • To extract DNA: – Punch a hole in the FTA paper – Put punch into tube and wash with solvents that isolate DNA from anything else – Punch itself is put into PCR reaction directly Differential Extraction:  • Add SDS and proteinase K • This combination will break apart the epithelial cells  • Releasing the female DNA into solution • Centrifuge to separate DNA from whole sperm cells – Female DNA • Then add SDS, proteinase K and DTT to solid pellet • DTT breaks apart sperm heads releasing DNA into solution – Male DNA DNA Quantification • Must be human DNA only • Must be in good enough shape for PCR results to be accepted and trusted Too much DNA: • Split/wide peaks • Leaking into other colors/lanes • Peaks that are off the scale Too little DNA: • Allele “drop­out”  Can be allele specific – so you will get the wrong genotype without knowing it Methods: Absorbance at DNA’s wavelength – Not as reliable or consistent – Uses too much of the sample Commonly used for Forensics: • Slot blot • Florescence based approaches/kits • Real­Time PCR – Quantitative PCR reaction Case: Victim’s Evidence • Man’s dead body is found stuffed into the trunk of a van • Examined the apartment of a suspect who had threatened the victim’s life before • Found bloodstains on: – Bedspread, Carpet and tennis shoe • Ran RFLP analysis to get a DNA profile Genotyping  1. RFLP Restriction Fragment Length Polymorphism  • Use a restriction enzyme  – A protein that cuts DNA based on sequence • Cut the region of DNA that is polymorphic • Restriction cut site has to be changed by the polymorphism in sequence • Enzyme will cut one allele and not another allele – Therefore different sizes of DNA strands • Two different sequences in DNA: ATGGGCTA  ATGTGTCTA TACCCGAT  TACACAGAT • Enzyme can cut one version, not other Therefore:  Allele 1 = two small pieces         Allele 2 = 1 large piece • Cannot be automated • Do not work well on degraded and contaminated DNA • Case: Quintanilla –  • two women were abducted and raped • Both women described similar men • Police compared DNA analysis between two cases and proved that both rapes were conducted by the  same man • One victim identified a man three times • DNA evidence did not match the man identified • Armando Quintanilla was arrested for a separate attempted assault • His DNA was taken  STRs: Short Tandem Repeat • 2, 3 or 4 bases repeated many times • Highly polymorphic – Often 8 or more alleles per region • Easily genotyped • Easily automated • Can be “multiplexed” – genotype more than one STR within same tube • Caused by different number of copies that people carry of a certain repeat:  ACTGACTGACTGACTG 4 copies ACTGACTGACTG 3 copies PCR:  1. Separate the Double Helix 2. Bind primers (2) to sequence you want to replicate 3. DNA Polymerase copies between two primers 4. Rinse and Repeat 5. Copies DNA between two primers exponentially  Running a Gel Electrophoresis: 1. PCR your DNA sample 2. Put the PCR products in a gel 3. Add electricity to the gel • Gel will separate the PCR products based on the size • As the products move through the gel’s matrix (pulled by neg. charge on DNA) • Smaller pieces will move faster • Once you have “run” the gel • Then you visualize the DNA within the gel in some manner (Photos, fluorescent dyes, radioactivity  labels DNA) Comparison STR & RFLP RFLP: • Cannot be automated • 1 to 8 weeks STRs: • 50 ng of DNA • 1 day • DNA cannot be contaminated • < 1 ng of DNA • DNA cannot be degraded • DNA can be contaminated • DNA can be degraded • Can be automated                          Amelogenin • Gene on X chromosome • But also on the part of the Y chromosome that is homologous to X chromosome: – PseudoAutosomal Region (PAR) • Therefore this gene is actually on both X and Y chromosomes • X chromosome has 6 bp deletion and Y chromosome doesn’t • Therefore XX genotype will be homozygous  • Identify (chromosomal) females • XY genotype will be heterozygous • Identify (chromosomal) males Y STRs • STRs (markers) that exist only on Y chromosome • Excellent for separately genotyping male and female mixed samples • Except show paternal inheritance only: • Always inherit the Y chromosome from father • Only have one copy + always the same Why the Y?  1. Sexual assault cases 2. Absence of sperm may make it difficult to separate male DNA from female DNA 3. May be overwhelming amount of female DNA – cannot read male genotype 4. Identifying more than one male from single mixed sample Genotyping mitochondria: • Certain conditions require genotyping the DNA within the mitochondria (mtDNA) • mtDNA is more stable over time/conditions • Can get more DNA – If sample/source is limited • Can get DNA from highly degraded source • May be the only DNA present in: – Bone, hair, fingernails and teeth • All cytoplasm comes from the egg or ovum • Sperm donates only nucleus to zygote • Therefore, all mitochondria are inherited from mother only Nuclear DNA: • Unique to every person • Recombines from parents to children Mitochondrial DNA: • Comes only from the ovum/egg • Without recombination • Therefore, is shared among family members – all maternal relatives Pros and Cons mtDNA Pros: Cons: • Stable, even under poor conditions • Shared among relatives • Multiple copies of mtDNA/cell • More time consuming to sequence • May be only option: bones, teeth and hair • Not present in database Marker = polymorphism in DNA that carries many alleles, does not affect phenotype Genotype = combination of alleles individual is carrying for one marker DNA Profile = combination of genotypes obtained for multiple markers  inclusion – if the DNA profiles match – Probability of seeing this match at random is calculated – This probability is used to measure how “damaging” evidence is Exclusion – if the DNA profiles fail to match – “Failure to match” could be lack of quality DNA, poor technology – Or because different source of DNA Case: Bungled Burglary  • Man throws a rock through a window of a bank • He then climbed into the bank and tried to rob it • He could not get any money or valuables • He went straight to the police station and turned himself in • DNA analysis was a match Degraded and Contaminated DNA Samples • Degrading DNA: • Any markers that did not work – – Dirty water regenotype with miniSTRs – Nucleases • If you still need more information – Sunlight from DNA then: – Microorganisms – Genotype with SNPs (single • Try genotyping with regular STRs nucleotide polymorphisms) – Mitochondrial DNA – Other methods • miniSTRs • Inverse relationship between size of PCR product and successful genotyping of degraded DNA – why? • Redesign the primers to generate smaller PCR products • “miniSTRs” • Moved the primers as close as possible to the repeat region • Contamination • Three primary sources: 1. Contamination that exists in the environment that has nothing to do with the crime  Control for this with:  Negative controls in PCR  Reference samples  If DNA is from people who live in area  Staff Elimination databases  If sample is from investigators analyzing DNA • 2. Contamination between two samples during analysis 3. Contamination of PCR reaction with PCR product from previous reaction • Both of these are controlled by: • Appropriate lab procedures • Removing PCR products away from PCR reaction set-up • Negative controls • Avoiding Contamination • Staff Elimination database – All people who ever come in contact with sample – Police, Analysts, etc • Reference samples • Using robotics for PCR automation • Negative controls in every set of samples • Well maintained laboratory procedures • Mixed Samples • Mixture arises when two or more individuals contribute to DNA sample during the crime • More markers used • More polymorphic loci • More chance that both samples will be heterozygous – see 3 or 4 peaks • Four Peaks • Both samples are heterozygous • Three Peaks • One sample is homozygous or alleles overlap • Two Peaks • Both samples are homozygous or alleles overlap • One Peak • Both samples are homozygous with exact same allele • Avoiding Mixtures 1. There is often multiple pieces of evidence from the same crime scene • Use a sample that is not mixed 2. Use differential extraction in sexual assault cases to fully separate out female vs. male DNA samples • Not enough DNA in sample • Final problem that arises with DNA analysis is not having enough DNA to get a successful PCR product • May only be one small spot of blood • Can collect reference samples but cannot get any more DNA from crime scene • 1. Use forensic evidence from another part of the crime scene instead • 2. Try to determine a DNA profile from the small amount of DNA that you do have • Ability to obtain a DNA profile from an extremely small amount of DNA • Less than 100 pg • Methods: – Increase number of PCR cycles – Amplify DNA first – then PCR – Decrease amount of volume of PCR reaction – Adding more PCR product to analysis – Changing analysis conditions • Analyst must work with extreme caution not to contaminate sample • Everything done in duplicate/triplicate • Negative controls are mandatory – Throw out any experiments with bands in negative controls • If suspect’s DNA matches – further experimentation must be done before evidence presented to jury • A Match? 1. Inclusion – if the DNA profiles match – Then we need to calculate the probability of seeing this match at random 2. Exclusion – non-match – The profiles are too different to possibly be the same individual 3. Inconclusive - unknown – There is not enough data to determine • Genotype AA = p Genotype Aa • Genotype aa = q 2 = 2pq • • • •


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