1. JS 115 Exam Prep Ch. 3: Butler (pgs 33-56) Ch. 6: Inman (pgs 65-95) Lay Saechao Annette Carmona Emily Saechao Jacob Levine Ruby Lo Kristin Lee-Janiak

2. Ch.3 Butler (pgs 33-42) DNA can come from a wide range of places: DNA can come from a wide range of places: blood, semen, bones, teeth, hair with root, saliva, urine, feces, etc. biological evidence blood, semen, bones, teeth, hair with root, saliva, urine, feces, etc. biological evidence Used to exclude or affiliate a person from involvement in a crime. Used to exclude or affiliate a person from involvement in a crime. DNA can be transferred to an individual or object from a victim, suspect, or witness in many ways. DNA can be transferred to an individual or object from a victim, suspect, or witness in many ways. It can be transferred through bodies touching or clothing. It can be transferred to an object such as a table, bed sheets, floor tiles or knife. It can be transferred through bodies touching or clothing. It can be transferred to an object such as a table, bed sheets, floor tiles or knife. Evidence should be collected, preserved, stored, and transported carefully before analyzing can begin. Evidence should be collected, preserved, stored, and transported carefully before analyzing can begin. When collecting the evidence, the person should make sure not to contaminate it. When collecting the evidence, the person should make sure not to contaminate it. Avoid contamination by wearing gloves when handling evidence, not sneezing or coughing around the evidence, air drying each piece and put into separate bags. Avoid contamination by wearing gloves when handling evidence, not sneezing or coughing around the evidence, air drying each piece and put into separate bags. Each bag should be marked with the case number, item number, date, and the person’s initials that is collecting the evidence. Each bag should be marked with the case number, item number, date, and the person’s initials that is collecting the evidence. When storing biological evidence, make sure it is stored in a dry and cold place to reduce bacterial growth or degradation of the DNA. When storing biological evidence, make sure it is stored in a dry and cold place to reduce bacterial growth or degradation of the DNA.

3. Ch.3 Butler (pgs 33-42) Reference samples Reference samples Used for comparing and excluding any family member or past boyfriends or girlfriends. Used for comparing and excluding any family member or past boyfriends or girlfriends. There are two ways to get a reference sample. There are two ways to get a reference sample. A blood withdrawal or a Buccal swab A blood withdrawal or a Buccal swab Presumptive tests Presumptive tests For identifying different biological fluids. For identifying different biological fluids. Used to see if the biological fluids are present or not on an item of evidence. Used to see if the biological fluids are present or not on an item of evidence. Presumptive tests for blood are immunochromatographic or luminal tests. Luminal is more popular for identifying the presences of blood. Presumptive tests for semen are the acid phosphatase (AP) or prostate specific antigen (PSA or P30) tests. It can also be Presumptive tests for blood are immunochromatographic or luminal tests. Luminal is more popular for identifying the presences of blood. Presumptive tests for semen are the acid phosphatase (AP) or prostate specific antigen (PSA or P30) tests. It can also be Identified by microscope. Saliva stains are virtually hard to see. There are two kinds of presumptive tests for detecting saliva- Phadebas and starch iodine radial diffusion test. Identified by microscope. Saliva stains are virtually hard to see. There are two kinds of presumptive tests for detecting saliva- Phadebas and starch iodine radial diffusion test.

4. Ch.3 Butler (pgs 42-50) 3 primary techniques used for DNA extraction: organic extraction, chelex extraction, and FTA paper. Organic extraction (also known as phenol-chloroform) Pro: Works well to produce high molecular weight DNA Con: Time consuming, involves the use of hazardous chemicals, requires the sample to be transferred between tubes increasing the risk of contamination. Chelex method More rapid than organic extraction involves fewer steps and fewer opportunities for contamination. FTA paper An absorbent cellulose-based paper Contains 4 chemicals that protect the DNA from degradation and preserving the paper from bacteria growth. Differential extraction Used by FBI and other forensic laboratories to isolate the female and male DNA for example in a rape case where they find both male and female DNA, they would use this method to separate the victims DNA from the perpetrator.

5. Ch.6: Inman Section 1&2 (pgs 65-71) Section 1: Isolation of DNA Chelex Extraction Chelex Extraction In cases where there’s only a little sample available (such as noticeable blood stains) The sample will be boiled with the chemical Chelex, Chelex job would be to separate any extra materials. DNA then would be released from the cells discharging the DNA needed. In cases where there’s only a little sample available (such as noticeable blood stains) The sample will be boiled with the chemical Chelex, Chelex job would be to separate any extra materials. DNA then would be released from the cells discharging the DNA needed. QiaAmp Extraction QiaAmp Extraction When the DNA undergoes some chemical washing off useless materials and then its drain, the clean DNA is then collected from the bottom in a drop of a liquid. When the DNA undergoes some chemical washing off useless materials and then its drain, the clean DNA is then collected from the bottom in a drop of a liquid. Organic Extraction Organic Extraction A method used to preserve the DNA in large pieces at the same time clean the DNA more carefully than the other methods. A method used to preserve the DNA in large pieces at the same time clean the DNA more carefully than the other methods. Samples will be cut into small pieces and soaked in warm solutions, gently releasing the cells from the substrates. Samples will be cut into small pieces and soaked in warm solutions, gently releasing the cells from the substrates. Then another chemical is used releasing the DNA removing the other components. The DNA then will be clean and turned into liquid to be used for RFLP or PCR based analysis. Then another chemical is used releasing the DNA removing the other components. The DNA then will be clean and turned into liquid to be used for RFLP or PCR based analysis. Differential Extraction Differential Extraction Type of procedure used to isolate DNA from a mixed sample of sperm and non sperm cells. Non sperm fraction is removed to a separate tube, while the sperm cells are treated with a few extra chemical to help remove them from the substrate. Type of procedure used to isolate DNA from a mixed sample of sperm and non sperm cells. Non sperm fraction is removed to a separate tube, while the sperm cells are treated with a few extra chemical to help remove them from the substrate. It is very important to determine how much human DNA there is and the quality of it to see if it’s ruined or not. It is very important to determine how much human DNA there is and the quality of it to see if it’s ruined or not.

6. Ch.6: Inman Section 1&2 (pgs 65-71) Section 2: Determining the Quality and Quantity of DNA Determination of Quantity Determination of Quantity For RFLP, “the quantitative information about total DNA is used to calculate how much restriction enzyme to add to each reaction… the proportion of human DNA determines how much sample to load on the analytical gel”. For RFLP, “the quantitative information about total DNA is used to calculate how much restriction enzyme to add to each reaction… the proportion of human DNA determines how much sample to load on the analytical gel”. For PCR the size of the DNA isn’t as important as how much human DNA is there, “a Slot Blot has been the method of choice to determine the amount of human DNA” The Slot Blot is when a small section of the sample is removed and tested, its then compared to known samples. For PCR the size of the DNA isn’t as important as how much human DNA is there, “a Slot Blot has been the method of choice to determine the amount of human DNA” The Slot Blot is when a small section of the sample is removed and tested, its then compared to known samples. Determination of Quality Determination of Quality Yield gel is used to determine the condition of the DNA, “it requires double- stranded DNA in high quantity for a result to be obtained.” Yield gel is used to determine the condition of the DNA, “it requires double- stranded DNA in high quantity for a result to be obtained.”

7. Ch.3 Butler (pgs 50-56) An important step before in depth analysis of a DNA sample begins is verifying that the sample that has been obtained is in fact human. An important step before in depth analysis of a DNA sample begins is verifying that the sample that has been obtained is in fact human. Determining the amount of usable DNA is also important because it will determine how many and what types of tests that can be completed. Determining the amount of usable DNA is also important because it will determine how many and what types of tests that can be completed. There are many different types of tests to quantify the amount of DNA present, ranging from tests that are cheap to expensive or from fast to slow. There are many different types of tests to quantify the amount of DNA present, ranging from tests that are cheap to expensive or from fast to slow. The most common type of test used for quantification is is the Slot Blot test. The most common type of test used for quantification is is the Slot Blot test. Works by capturing DNA on a nylon membrane and then comparing chemiluminescent signal intensities of the sample to a set of standards. Works by capturing DNA on a nylon membrane and then comparing chemiluminescent signal intensities of the sample to a set of standards. Can be done very quickly, or more time can be taken to get more sensitive results Can be done very quickly, or more time can be taken to get more sensitive results One limitation of the test is that it lacks the sensitivity required to conclusively determine that there isn't any DNA present. One limitation of the test is that it lacks the sensitivity required to conclusively determine that there isn't any DNA present.

8. Ch.3 Butler (pgs 50-56) Aluquant test works by probing Alu repeats in the human genome. Aluquant test works by probing Alu repeats in the human genome. This process that can be automated by robotics causes DNA to emit light, which is measured to establish how much DNA is present. This process that can be automated by robotics causes DNA to emit light, which is measured to establish how much DNA is present. A Real Time PCR test not only measures: A Real Time PCR test not only measures: the amount of DNA found but the usability of it for testing. the amount of DNA found but the usability of it for testing. The test measures quantity, quality, and tests for any inhibitors mixed with the sample. The test measures quantity, quality, and tests for any inhibitors mixed with the sample.

9. Ch.6: Inman Section 3&4 (pgs 71-77) Section 3: RFLP Analysis Restriction enzyme digestion and Southern blotting Restriction enzyme digestion and Southern blotting DNA, restriction enzyme, and other components DNA, restriction enzyme, and other components Digested DNA loaded on gel Digested DNA loaded on gel DNA fragments separated in electric field DNA fragments separated in electric field DNA fragments transferred to nylon membrane and ready for probing DNA fragments transferred to nylon membrane and ready for probing Probing and detection Probing and detection a. radioactive probe is applied to the membrane a. radioactive probe is applied to the membrane b. excess probe is removed b. excess probe is removed Probe hybridizes to specific DNA fragments Probe hybridizes to specific DNA fragments radioactive membrane exposed to x-ray film (autorad) radioactive membrane exposed to x-ray film (autorad) x-ray film ready for interpretation x-ray film ready for interpretation Analysis/Interpretation Analysis/Interpretation As many as 5 or 6 loci analyzed, so about 10 to 12 bands detected (1 or 2 bands at a time) As many as 5 or 6 loci analyzed, so about 10 to 12 bands detected (1 or 2 bands at a time) Each probe detects a different locus Each probe detects a different locus Once information from first probe is recorded, it is removed and the nylon membrane is exposed to the next probe in series. After second probe has been detected and recorded the process is repeated for every additional probe. The exposed films/autorads from each particular probe provide a permanent record of the results of the analysis. Once information from first probe is recorded, it is removed and the nylon membrane is exposed to the next probe in series. After second probe has been detected and recorded the process is repeated for every additional probe. The exposed films/autorads from each particular probe provide a permanent record of the results of the analysis. Samples that look locations are compared from lane to lane to identify any similar patterns are then subjected to computer imaging and analysis. Samples that look locations are compared from lane to lane to identify any similar patterns are then subjected to computer imaging and analysis.

10. Ch.6: Inman Section 3&4 (pgs 71-77) Section 4: PCR Amplification PCR Amplification often performed on samples that are deemed too minuscule or highly degraded to give a reliable RFLP results. PCR Amplification often performed on samples that are deemed too minuscule or highly degraded to give a reliable RFLP results. Process replicates segments of DNA millions of times and is dependent on the Taq polymerase enzyme Process replicates segments of DNA millions of times and is dependent on the Taq polymerase enzyme Can survive high temperatures Can survive high temperatures 3 Main Steps 3 Main Steps Denaturation – separate 2 strands of DNA so that each stand can be used as a template for synthesis of a new strand. Denaturation – separate 2 strands of DNA so that each stand can be used as a template for synthesis of a new strand. Annealing – involves annealing of DNA primers Annealing – involves annealing of DNA primers 2 different primers define the end-point of a particular segment that is amplified, one at each end 2 different primers define the end-point of a particular segment that is amplified, one at each end Extension – raw materials of DNA are hooked together by Taq polymerase to create new DNA strands Extension – raw materials of DNA are hooked together by Taq polymerase to create new DNA strands new strand manufactured, base by base, with a sequence complementary to the beginning template new strand manufactured, base by base, with a sequence complementary to the beginning template at the end of the first cycle of PCR, the segment of interest has been duplicated. at the end of the first cycle of PCR, the segment of interest has been duplicated. The 3 steps are repeated over and over again, each time doubling the number of copies of DNA The 3 steps are repeated over and over again, each time doubling the number of copies of DNA A product gel is run using a small portion of the sample to check that the PCR reaction has been successful A product gel is run using a small portion of the sample to check that the PCR reaction has been successful

11. Ch.6: Inman Section 5&6 (pgs 77-95) Section 5: Analysis of PCR Product PCR product (the product of PCR reactions) PCR product (the product of PCR reactions) Analyzed in one of two ways Analyzed in one of two ways Sequence polymorphisms (AmpliTypePM+DQA1& Mitochondrial DNA) Sequence polymorphisms (AmpliTypePM+DQA1& Mitochondrial DNA) Length Polymorphisms (D1S80, STRs, Gender ID) Length Polymorphisms (D1S80, STRs, Gender ID) AmpliTypePM+DQA1 AmpliTypePM+DQA1 Reverse dot blot format Reverse dot blot format Mitochondrial DNA Mitochondrial DNA Four different tubes color coded tubes Four different tubes color coded tubes Final computer output shows overlapping colored peaks, making where each base ends Final computer output shows overlapping colored peaks, making where each base ends Identification of mutational hot spots Identification of mutational hot spots Based on same technology as reverse dot blot Based on same technology as reverse dot blot When hybridization with probe occurs, area turns blue When hybridization with probe occurs, area turns blue Read by noting which part of the strip matches defined alleles Read by noting which part of the strip matches defined alleles Length Polymorphisms Length Polymorphisms Most common in forensic DNA testing Most common in forensic DNA testing PCR product loaded into a gel PCR product loaded into a gel See DNA bands using a silver stain See DNA bands using a silver stain Compare bands Compare bands Analyzed using fluorescent detection and automated analysis Analyzed using fluorescent detection and automated analysis

12. Ch.6: Inman Section 5&6 (pgs 77-95) Section 6: Automated Analysis Systems Ideal for labs that have samples with the same repetitive handling process or for labs with a higher throughput of case samples Ideal for labs that have samples with the same repetitive handling process or for labs with a higher throughput of case samples Automated DNA Extraction and Amplification Automated DNA Extraction and Amplification Liquid Blood Transfer Liquid Blood Transfer DNA Extract DNA Extract Quantitation Quantitation Amplification Amplification Issues Issues Well-to-well carryover (contamination) Well-to-well carryover (contamination) Bar code reader  small error rate Bar code reader  small error rate Trasnfer errors Trasnfer errors Random quality control checks to ensure equipment is working properly Random quality control checks to ensure equipment is working properly Automated systems do not eliminate the human Automated systems do not eliminate the human