Unit 2- DNA Analysis

Transcription

1 Unit 2- DNA Analysis

2 Discovery of DNA structure 1950 s Rosalind Franklin & Maurice Wilkins photograph DNA using x-ray diffraction 1

3 Discovery of DNA structure 1953 James Watson & Francis Crick develop a model of a DNA molecule 2

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5 DNA = Deoxyribonucleic Acid DNA is located in the nucleus & in the mitochondria of cells DNA is the code of genetic information that tells cells how and when to make proteins Proteins make you unique = nose s, toes, eyes, hair, your enzymes, etc. 4

6 Organization of DNA in cell Nucleus has 23 pairs of Chromosomes (46 total from parents) Chromosomes are wound up strands of DNA DNA sections that code for traits (aka proteins) are called genes 5

7 General Structure of DNA Double helix two coiled DNA strands Composed of nucleotides pieces connected together Four bases make the rungs: Adenine Cytosine Guanine Thymine Bases always pair A to T and G to C (complementary base pairing) In humans, the order of these bases is 99.9% the same. 6

8 Karyotype- picture of chromosomes 7

9 Human Genome Project ( ) Goals: identify genes in human DNA (20,000 +) determine the sequences of ATCG s that make up human DNA store this information in databases SPECIAL NOTE: DNA Analysis DOES NOT reveal personal information about a suspect It cannot tell race, eye color, hair color, etc not possible yet! 8

10 Bodily sources of DNA All Cells that have a nucleus: white blood cells (NOT RED BLOOD Cells- they don t have a nucleus) Semen saliva contains buccal (cheek cells) hair root teeth bone Any tissue All cells contains thousands of mitochondria which contain maternal DNA 9

11 Uses of DNA Profiling identify potential suspects exonerate individuals identify crime and casualty victims (9/11) establish paternity match organ donors 10

12 Milestones in DNA Analysis Alec Jeffreys 1985 isolated DNA markers and called them DNA fingerprints RFLP technologies used mid 1990 s Kary Mullis 1985 developed PCR testing (perfected by the mid 1990 s) DNA evidence introduced for the first time in court 1988 FBI starts DNA casework 1991 first STR paper 1998 FBI launches CODIS database 11

13 Coding vs. Non- Coding sections of DNA Coding DNA- 5 % of your DNA recipe that CODES for proteins that make you unique Junk DNA/ Non Coding DNA 95 % of DNA it doesn t seem to code for anything (yet!). repeats the same base pair sequence over and over (aka Tandem Repeats) Best for I.D ing people forensically (b/c varies most in people) 12

14 Reading DNA fingerprints It s Dino s because it matches crime scene. Bugsy has some DNA strands that aren t in crime scene sample 13

15 Sometimes DNA is mixed at a crime scene Miss Scarlet & Miss White each contributed ½ the DNA to the scene. 14

16 Paternity Case 2paternity cases below (remember kids get ½ their DNA from each parent): Usually use terms: EXCLUDED or INCLUDED 15

17 DNA Typing (Fingerprinting/ profiling/ analysis) 3 main technologies have been used: 1) RFLP Restriction Fragment Length Polymorphisms - Developed in 1985 & used until mid 1990 s 2) PCR Polymerase Chain Reaction - Developed in the 1980 s, but perfected in mid 1990 s - Still used today as a step in the STR process 3) STR- Short Tandem Repeats - Developed in the 1990 s - Used almost exclusively today 16

18 The RFLP Technique Restriction Fragment Length Polymorphisms Polymorphisms = Variations in DNA sequence between individuals General Overview: 1) Isolate separate DNA from the cell of desired individuals & evidence 2) Cut using restriction enzymes to cut DNA into smaller fragments 3) Separate/ Sort by size using electrophoresis which creates banding pattern. 4) Analyze the specific allele patterns for identification DNA Fingerprint 17

19 1. DNA is Isolated/ Extracted 3 Steps in a DNA extraction: a) Break open cells by grinding & remove membrane lipids by adding a detergent. b) Remove proteins bound to the DNA, by adding salt. c) Precipitate DNA in cold alcohol (DNA is insoluble in alcohol and clings together) This bead beater is used in the breaking apart or "lysing" of cells DNA 18

20 2. Restriction enzymes cut DNA into fragments Restriction Enzymes act as molecular scissors that cut DNA into pieces. Come from bacterial cells where they were discovered Each enzyme has a specific DNA sequence it will look for and cut. Everyone has a lot of repeating DNA codes that are unique (tandem repeats) Enzymes cut at these unique sections resulting in everyone having different fragments of DNA. (see image) 19

21 3. Gel electrophoresis- Separate fragments based on size DNA is loaded into wells inside a gel An electrical current is moved through a gel (DNA is negatively charged so it will move toward the positive side of the gel box) molecules to sort by size. The smaller, lighter molecules will move the furthest on the gel, creating a banding pattern 20

22 Electrophoresis 2. Turn on electrical supply to move DNA 1. Pipette & load DNA into wells in the gel. 3. See DNA run through gel thanks to the electrical current larger fragment= slow Small fragment= fast 21

23 4. Radioactive markers are added to bond to the DNA to make it visible. Gels are read 22

24 Three Possible Outcomes Match The DNA profile appears the same. Lab will determine the frequency. Excluded The two samples originate from different sources. Inconclusive The data does not support a conclusion as to whether the profiles match. 23

25 Disadvantages of RFLP Fingerprinting Requires lots of long pieces of DNA Isssue: longer pieces of DNA tend to degrade and break into smaller pieces, randomly. Crime scene DNA is often in adverse conditions. 24

26 DNA Profiling Casewho s the dad? Infertile couple she cannot conceive but he was a sperm donor Surrogate s husband (is it his baby?) (is it his baby?) Surrogate mother (her egg) 25

27 DNA profiling Case- #2 Joe Millionaire & potential father Parents of father Mother claiming infant son was fathered by deceased Joe Millionaire 26

28 DNA Replication = copying 1 DNA molecule into 2 identical DNA molecules Helicase = enzyme that unzips (breaks H Bonds) the strands of DNA. DNA Polymerase = enzyme that attaches new complimentary bases on the unzipped strands Semi-conservative Replication = new molecules are ½ new & ½ old strands 27

29 PCR Polymerase Chain Reaction technique used for making copies of a DNA molecule valuable when the amount of evidence is minimal Millions of copies of DNA can be made from a single speck of blood Stems from knowledge of how DNA replicates itself Thermocycler- machine used to do PCR 28

30 1) Heat DNA strands =strands separate (unzip). 2) Cool the mixture and add a primer, a short sequence of base pairs, that will add to its complementary sequence on the DNA strand. 2) Add a DNA polymerase and a mixture of free nucleotides to the separated strands. DNA will re-build. Heat again to around 75 C for the completion. PCR Procedure Every cycle the DNA amount is doubled. 29

31 Advantages of PCR Typically PCR cycles are run resulting in billions of copies of DNA (mathematically ) Each cycle that doubles the DNA takes about 2 minutes. Minute amounts of DNA may be used for amplification (less than 1 billionth of a gram). Can get enough DNA from envelopes, stamps, soda cans, & cigarette butts to run PCR 30

32 STR- Short Tandem Repeats Current method of choice for DNA typing. The basics Example of STRs in a person STR s = short sequences of 2 to 5 bases that repeat themselves ( x s) 6 repeats from 1 parent RFLP s (older technique) are base pairs long and can repeat up to 1000 times. 8 repeats from 1 parent This is the TH01 loci for an individual See related internet links on class website for STR demonstration. 31

33 By continuing the process with additional STRs from other genes, you can narrow down the probability of DNA belonging to only one person. Here s all the ones that are used: 32

34 Short Tandem Repeats (STR) STR typing is visualized by peaks shown on a graph. Each represents the size of the DNA fragment. The possible alleles are numbered for each loci. Norma's genotype is 15 repeats, 15 repeats at the locus D3S1358, 14 repeats, 16 repeats at vwa, 24 repeats, 25 repeats at FGA (gets on from each parent) 33

35 EX: STRS Paternity Profile Great website: linformation.xsp 34

36 Using STR DNA Profiling Results David & Karen are parents of a missing child: DNA Profile from remains found in a shallow grave: Could this be their child? No, the DNA for vwa & FGA doesn t show ½ from each parent 35

37 Short Tandem Repeats (STR) Procedure Extract the gene TH01 from the sample. (TH01 has seven human variants with a repeating sequence of A-A-T-G) Amplify the sample by means of PCR Separate by electrophoresis Examine the distance the STR migrates to determine the number of times TH01 repeats Each person has two STR types for TH01 one inherited from each parent This is the TH01 loci for an individual: Received 6 repeats from one parent and 8 repeats from the other. 36

38 Advantages of STR Combines the strengths of PCR and RFLP while minimizing the weaknesses: requires small amount of DNA- need only 18 DNA bearing cells. 1 billionth of a gram (1/100 th needed for RFLP). STR s found in great abundance in human genome so allow for greater discrimination in results (1 in 1 trillion people for some odds) DNA is less susceptible to degradation because of the small amount needed. Requires less time to run 37

39 DNA Identification Based on Probability probability of a person matching a random DNA sample at any 1 STRS site is roughly 1/10 3 STRS sites? 1/10 x 1/10 x 1/10 = 1/1000 all 13 STRS sites would mean that the chances of matching a random DNA sample are about 1 in 10 trillion: 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x 1/10 x = 1/10,000,000,000,000 probability of two different people matching at all 13 STRS sites is virtually zero. 38

40 Types of DNA Mitochondrial found in the cytoplasm inherited only from mother each cell contains 100 s s of mitochondria used when nuclear DNA typing is not possible (old, degraded samples) More costly, time consuming to test Nuclear found in the nucleus inherited from both parents each cell contains only 1 nuclei 39

41 FBI s CODIS DNA Database Combined DNA Index System (1998) Computer system has: Convicted Offenders DNA Crime Scene DNA Arrestee DNA Unidentified/ Missing person s DNA System can cross reference info. from all 50 states Prop 69 in CA 40

42 Collection and Preservation of Biological Samples Photograph objects containing samples Limit personal contact & change gloves often All clothing should go to lab, even if blood is not seen Package items separately in breathable container (not plastic!) blood is allowed to dry first Refrigerate evidence until delivered to a lab 41

43 Evidence baseball bat or similar weapon hat, bandanna, or mask eyeglasses facial tissue, cotton swab dirty laundry Toothpick, cigarette, stamp or envelope, bite mark tape or ligature bottle, can, or glass used condom blanket, pillow, sheet through and through bullet Fingernail (or partial) Source of DNA sweat, skin, blood, tissue sweat, hair, dandruff sweat, skin mucus, blood, sweat, semen, ear wax blood, sweat, semen saliva skin, sweat saliva, sweat semen, vaginal/ rectal cells sweat, hair, semen, urine, saliva blood, tissue blood, sweat, tissue 42

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45 Cover Photo Controversy

46 Time was forced to recall the original cover story (left) and replace it with the one on the right.

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