General DNA Information

Transcription

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2 Use of this PPT This PPT has lots of information. I may or may not discuss each slide. Use the information to answer questions in your Question Packets. The packets are the study guide for the tests (there are two tests to this unit). Any slide with a * in the title or text, is important to use when studying for the test. 2

3 General DNA Information James Watson and Francis Crick discovered the structure of the DNA molecule Double helix--two coiled DNA strands Composed of nucleotides that are named by the nitrogen base they contain: Adenine (A)-Thymine (T) * Cytosine (C)-Guanine (G) * HHMI DNA Strands 3

4 C G A G T C Numerous hydrogen bonds join the two DNA strands in the center of the molecule, holding it together. * 4

5 *Two Types of DNA * Nuclear DNA-(nucDNA) found in the nucleus of the cell-may individualize to a person Mitochondrial DNA-(mt DNA) found in the mitochondria of the cell-only class evidence. Both types of DNA above may be used to identify the source of evidence. 5

6 Mitochondrial DNA Class evidence only found in the mitochondriasite of cell respiration ATP DNA is circular in shape genes on DNA code for proteins used in cell respiration 1,000 s of mitochondria in each cell = 1,000 s of copies of mtdna in each cell (one in each mitochondrion) not as fragile as nuclear DNA--can be found in skeletal remains and hair inherited ONLY from mother 6

7 Mitochondria The mitochondria in the tail of the sperm fall off after the nucleus enters the egg. The only mtdna in the ovum is from the mother. 7

8 ndna genome: 30,000,000 base pairs 24,000 coding genes 97% non-coding variable regions mtdna genome: 17,000 base pairs 38 coding genes small variable regions 8

9 *Nuclear ndna* 46 chromosomes in each nucleus linear in shape (not circular) 23 pair of chromosomes (homologs) within the pair, one chromosome is maternally (M) inherited and the other is paternally inherited (P) M fragile, easily degraded contains 20-24,000 coding genes 3 billion base pairs A-T or C-G P 9

10 * Value of DNA evidence: * 1. Nucleotide sequences in ndna and mdna are identical in identical twins A DNA profile will NOT distinguish between them. It will, however, narrow the search to one of the two twins! 2. In most cases a ndna profile is considered individualized evidence. In the case of identical twins, it is the best class evidence. It usually has high associative and probative value. 3. mdna is considered good class evidence with lower AV and PV 10

11 * ndna: 23 Pair = 46 Linear pieces of DNA* You have TWO of each type of chromosome (M+P) in the nucleus of each cell. SEX CHROMOSOMES 11

12 *Human Genome* The genome is all of the genes in your DNA. The DNA in one cell includes: 3 billion base pairs (A-T s and C-G s) * 6 billion nucleotides Fortunately, we do not need that much ndna to produce a DNA profile. *All we need are 26 segments or alleles of non-coding DNA. * 12

13 *What is a Gene? * Segments of the DNA at a specific location. : Coding Genes carry the code for important proteins needed for survival. They don t differ much between people. Non-coding genes don t form proteins. Differ between people so can be used to produce a DNA profile to identify where evidence came from. You have two copies of each gene. One from your mother (maternal) and one from your father (paternal). These two copies may or may not be identical. They are called alleles. 13

14 *Mutations Cause Variation* In order to differentiate between two people, we will need to detect variations or differences between their alleles. These variations are caused by mutations. Mutations are accidental mistakes made during DNA replication. Since non-coding genes do not code for proteins, mutations in them are not harmful. They will be inherited and carried to the next generation and are BEST to use in a DNA profile! 14

15 *Non-Coding Regions of ndna * % of ndna is non-coding (does not contain a recipe or code for a protein). Variation (mutations) within these areas is high from person to person because it does not alter protein production and therefore, does not affect the survival of the person. Scientists use sequences within the noncoding regions to generate a DNA profile because they have high variation between individuals. 15

16 *Alleles on Chromosomes* Allele-a sequence of nucleotides at a gene locus. You have two alleles at the same gene locus or location on two homologous chromosomes. M P If the allele segments are the same they are considered homozygous. If they are different they are heterozygous. 16

17 *GOAL: DNA Profile * (aka. DNA fingerprinting) Term used to describe the genotypic results determined from the visualization (banding pattern) of 13 specific loci or gene locations from nuclear, NON-CODING DNA. (26 total segments.) Gel Electrophoresismethod used to visualize the alleles. 17

18 Where are the variations in DNA? Within coding genes Between coding genes 18

19 * Short Tandem Repeats-STR s* Some of the non-coding DNA regions have repeat regions. These are particular sequences may be repeated 1-30 times. They are called tandem repeats: CATTA CATTA CATTA CATTA The shorter, 3-7 nucleotide repeats used in DNA profiling are called STR s. STR stands for short tandem repeats In a DNA profile, you want to visualize or identify about 13 different STR s. 19

20 Example: Repeat sequence Note: Most STR s are 3-7 repeats in length. 20

21 Consider this example from ndna profiles: If Malia has 3 repeats of CATTA at one gene locus and Sasha has 5 repeats of CATTA at the same gene locus then, the Obama siblings will have different ndna profiles! 21

22 *Visualizing STR s produces a DNA Profile * Each individual has a particular number of STR repeats. Some have many repeats, others may only have a few repeats. If we visualize the lengths of several of these regions, called TARGET SEQUENCES, we can produce a DNA PROFILE. 22

23 *Steps in Building a DNA Profile* 1. Extract and Purify DNA 2. Amplify Target Sequences PCR 3. Visualize Target Sequences- Gel Electrophoresis 4. Calculate the Probability of the Match 23

24 *Finding sources of DNA * Can be found in body cells, tissues and in most body fluids and eliminations. (saliva, semen, blood, urine, feces) Police collect samples from buccal (cheek) cells DNA obtained from blood comes from white blood cells (red blood cells do not have a nucleus or mitochondria)

25 *Step I-Extraction and Purification of DNA * DNA in the sample is isolated and cleaned to exclude all materials other than cells. A mixture of organic chemicals is used to separate the DNA from other cellular components (membrane, organelles, etc.) It is simple, you will do this in class!

26 *Step II-Amplification of DNA* Many times, the biological evidence at a crime scene is scant. Polymerase chain reaction: PCR Method to amplify or make multiple copies of the DNA of interest. Otherwise, since DNA is so small, you would not be able to see or visualize the results.

27 Step II-PCR amplification is similar to the replication of DNA that takes place inside the nucleus of a cell before cell division. The difference is that we only amplify small, target segments of the DNA that hold the STR s.* 27

28 StepII: Amplify Target Sequences using PCR Method: 1. Heat the DNA strands to separate the strands(unzip). 2. Add primers and nucleotides to make new strands. 3. Replication of the target sequence will occur. 4. Cool down to 72 degrees to stabilize the new strands. 5. Do this several times to make more and more Target sequences. 28

29 Step II-PCR continued cycles, as many as a billion copies of the target sequence are produced from a single starting molecule. 7. Quick: each cycle takes less than two minutes from start to finish. 8. The resulting DNA can undergo gel electrophoresis for visualization. PCR Animation sumanas inc. DNALC animation site for PCR 29

30 DNA Amplification with the Polymerase Chain Reaction (PCR) Starting DNA Template Separate strands (denature) Forward primer 5 3 Add primers (anneal) Make copies (extend primers) 3 5 Reverse primer 30

31 PCR Copies DNA Exponentially through Multiple Thermal Cycles Original DNA target region Thermal cycle In 32 cycles at 100% efficiency, 1.07 billion copies of targeted DNA region are created 31

32 **STR Target Sequences Produced by PCR** AATG 7 repeats 8 repeats the repeat region is variable between samples while the flanking regions where PCR primers bind are constant Homozygote = both alleles are the same length because they have the same number of repeats. Heterozygote = alleles differ in number of repeats and length.

33 *Thermocycler* tool used to conduct PCR. holds numerous samples in small test tubes under top lid. automatically warms and cools samples. amplification takes place in microcentrifuge tubes. 33

34 *Advantage of Short Tandem Repeats-STR s and PCR* Small amounts of DNA can be amplified and analyzed from dental molds, cigarette butts, eating utensils, chewing gum, postage stamps, ski masks, licked envelopes, toothbrushes, razor shavings, bandaids and fingerprints (coroner touch DNA v2). 34

35 *3rd Step: Visualization of the Target Sequences using Gel Electrophoresis* Technique used to separate fragments of DNA into bands that can be visualized. Electrophoresis tray and power source 35

36 * The DNA has a negative charge! 36

37 *Banding Patterns* A LANE is the vertical path in which the DNA travels from the well to the positive end of the gel tray + 37

38 *Review: How bands form* STR target sequences are placed in wells at one end of an agar gel. A direct current is run through the gel. The DNA migrates or travels to the positively charged end of the gel. Smaller fragments travel more quickly than larger fragments. 38

39 *What is a band?* A band is a visual spot on the Gel. It is composed of 1,000 s of target sequences of the same length (same number of STR repeats, or base pairs). How did we get 1,000 s of these sequences? Can you identify a band? a lane? a heterozygous sample? 39

40 *How many bands in a lane?* If a person is heterozygous, their M and P alleles are different lengths. They have different numbers of the repeat motif. They will show two bands at different migration distances on the gel. If a person is homozygous, their alleles are the same length and the target sequences will travel the same distance showing only one band on the gel. 40

41 *Gel Rule* Bands that migrate or travel further from the wells contain target sequences that are SHORTER and have FEWER repeats. Bands that don t travel as far have more repeats. Analogous to paper chromatography. Animation of Gel Electrophoresis vs. Chromatography 41

42 Short Tandem Repeats STRs Alleles differ by the number of repeats. Largest AGCT AGCT AGCT AGCT AGCT AGCT AGCT AGCT AGCT AGCT 4 Repeats 3 Repeats 2 Repeats 1 Repeat Smallest

43 *How to determine a match:* If evidence (unknown) samples are run at the same time as suspect samples (known), and the bands appear at the same distance from the wells, the samples match. If unknown samples show migration at different distances compared to the known sample, the samples do not match. 43

44 Sample Results for STR D18: E S1 S2 S1 or S2=Known suspects E1= Unknown Evidence Which suspect (1 or 2) matches the unknown evidence? Is S2 homo or heterozygous for the D18 STR? 44

45 *How do you determine the genotype of a sample?* A LADDER is a sample containing DNA segments of known lengths. These samples are run at the same time as your known and unknown STR samples. Ladder 100 bp-> <-Samples-> 45

46 *How many repeats in a band?* Take the length of a band minus the number of nucleotides in the leading and lagging primers and divide by the number of nucleotides in the repeat. Ex. Length of band= 100bp Minus Leading primer= 27 bp Minus Lagging primer= 25 bp 48 bp Divide by 4 nucleotides per repeat = 12 In this case, if the sample has only one band, its genotype is 12,12 46

47 *Important to use all 13 STRs!* Just because you have determined a match between an unknown and known sample on the basis of one STR (ex. TPOX), it does not mean the two samples are a match. In order to use your results in court, you must run gel analysis of all 13 STR s to form a DNA profile and compare results of all 13 STR s to suggest a MATCH. 47

48 TPOX 13 CODIS Core STR Loci with Chromosomal Positions D3S1358 D5S818 FGA CSF1PO D8S1179 D7S820 TH01* VWA AMEL D13S317 D16S539 D18S51 D21S11 AMEL 48

49 *Example of Results:* DNA Profile STR Genotype CSF1PO 11,12 FGA 21,21 TH01 7,7 TPOX 8,8 VWA 16,17 D3S ,16 D5S818 11,12 D7S820 10,10 D8S ,13 D13S317 11,11 D16S539 11,12 D18S51 15,15 D21S11 30,30 A DNA profile will list the number of repeats or the GENOTYPE for each of the 13 STR s. If the genotypes of the unknown evidence are identical to the genotypes of a known suspect, it is considered a match. 49

50 ***Three Possible Outcomes*** Match--The unknown and known DNA genotype profiles appear the same. A Profile Probability must be calculated. Exclusion The comparison shows differences that can only be explained by the two samples originating from different sources. Inconclusive The data does not support a conclusion as to whether the profiles match. Perhaps the sample has degraded over time? 50

51 DNA can be legally collected from: All convicted felons in NJ back to July 1995 Anyone under criminal arrest by federal authorities NJ-persons suspected of certain criminal acts. Illegal immigrants detained by federal authorities In England, all criminal suspects! 51

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53 Practical Applications of DNA Profiling 1. Paternity Testing * 2. Forensics:Matching Suspect with* Evidence (2 out of 3 are rape cases) 3. Personal Identification 4. Historical Investigations 5. Missing Persons 6. Mass Disasters 7. Military Dog Tag 8. Convicted Felon Databases CODIS* 53

54 *FBI s CODIS DNA Database* Combined DNA Index System Started by FBI Launched October 1998 Used for linking serial crimes and unsolved cases with repeat offenders Links all 50 states Requires 13 core STR markers

55 DNA--The Search for Innocence DNA implicates the guilty and exonerates the innocent--r. Saferstein Over the years many innocent people have been wrongly convicted with no way of proving their innocence. Since the late 1980 s, DNA technology has been available. People, as a result, have been reopening cases and testing the evidence for DNA. Some have been exonerated because their DNA did not match. Innocence Project: Group of college law students work on cases where DNA may prove innocence (or guilt) 55

56 *Multiplex PCR* All 13 STR s can be copied at once Need only small amounts of DNA Can handle comingled and degraded samples Fluorescent dyes used to distinguish different STR alleles

57 ABI PRISM 3700 DNA Analyzer Multiplexing--technique in which the number of repeats can be determined for more than one STR at a time. Fluorescent tags are added to each target sequence so it can be identified. 57

58 capillary Syringe with polymer solution Injection electrode Outlet buffer Autosampler tray Inlet buffer 58

59 Close-up of ABI Prism 310 Sample Loading Area Electrode Capillary Sample Vials Autosampler Tray 59

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61 *Multiplex Computer Results* AmpFlSTR SGM Plus kit AM D19 D3 D8 TH01 VWA D21 FGA DNA Size (base pairs) D16 D18 D2 AM D19 D3 D8 TH01 VWA Results are from two different individuals above and below. D21 FGA D16 D18 D2 Simultaneous Analysis of 10 STRs and Gender ID 61

62 *Probability of the Match* In order to be accepted into a court of law: The genotypes for all 13 alleles must match! Still, if they do match, that is not enough. The results of a DNA profile must be analyzed to determine the frequency or probability of any random person having the same alleles or genotype. If the probability of having the STR repeats is LOW, the evidence has better value because it will be LESS likely to find a random person with the same genotypes. 62

63 *Determining Probability* Databases have been established that show the probability of an individual having a particular STR repeat. For example, the probability of having 7 repeats of the TH01 allele is approximately 18% for the Caucasian population. That means that 18% of the Caucasian population has 7 repeats of the TH01 allele. Another way of saying this is that 18 of 100 Caucasians sampled will have 7 repeats of the TH01 allele. 63

64 Frequency STR allele frequencies based on cultural background Number of repeats TH01 Marker Caucasians (N=427) Blacks (N=414) Hispanics (N=414) *Proc. Int. Sym. Hum. ID (Promega) 1997, p

65 *DNA Profile Probability* The probability that any random individual will have a specific set of STR repeats over 13 different markers (26 alleles). Calculated by multiplying the probabilities of each STR marker. If you find a match between evidence and a suspect, the smaller the Profile Probability, the greater the chance that you have found your true match to the evidence! 65

66 *Calculating a Profile Probability* A piece of DNA evidence matches a suspect based on 4 STR s that have the frequency of 5, 8, 10 and 3 percent. What is the probability that a random person might also match these 4 markers? or.0012% of the population Assume 300 million people in the U.S. 1 in 3600 people would have all 4 markers. Is this good evidence? 66

67 *Probabilities of having more than one STR in common:* Product rule--used to determine the probability of having all 13 different sets of STR repeats. If the probability of having a 3,4 repeat for D7S820 is 5% and the probability of having a 6,9 repeat for D21S11 is 3 %, then the probability of having both sets of repeats is only.03 X.05 =.0015 or.15%. The probability of having 13 sets of STR s ends up being a VERY small number. (ex. 2 X ) That is less than one in 6 billion. 67

68 The best probability for evidence? 1,000, million 1,000,000, billion 1,000,000,000, trillion How many people are there living on earth? world population counter If only one in 7 billion people is likely to have the 26 STR repeats, the evidence is superior the match is valid.

69 *Paternity Results* Paternity matches are based on the fact that a child will inherit one allele from its mother and one from its father. In order for a man to not be excluded as father of the child, he must have the one, obligate paternal allele not contributed by the mother. For example, if the child has 6,7 for the FGA STR, and the mother is a 5,6 the father must have a 7 repeat on at least one of his FGA alleles. 69

70 Paternity Results Note: We will not be calculating the parentage index in this class, but the higher the number, the less common the allele and the more likely this man contributed the obligate paternal allele. 70

71 Historical Information-FYI 1800 s Fredrich Meichner discovered DNA 1953 Watson, Crick and Wilkins received Nobel Prize for structure of DNA 1985 Alec Jeffreys isolated DNA markers and called them DNA fingerprints 1985 Kary Mullis developed PCR testing FBI starts DNA casework first STR research published FBI launches CODIS database. 71

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