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“The capacity to blunder slightly is the real marvel of DNA. Without this special attribute, we would still be anaerobic bacteria and there would be no music.” —Lewis Thomas, Physician, author DNA Analysis Students will learn: That DNA is a long-chain polymer found in nucleated cells, which contains genetic information. That DNA can be used to identify or clear potential suspects in crimes. How DNA is extracted and characterized. How to apply the concepts of PCR and STRs to characterize DNA. The role that statistics plays in determining the probability that two people would share a DNA profile Chapter 11 Kendall/Hunt Publishing Company 1 DNA Analysis Students will be able to: Explain that DNA is a long molecule, tightly packed in the form of a chromosome with genetic material wrapped around it. Isolate and extract DNA from cells. Describe the PCR process Describe STR testing and calculate probabilities of identity using STR. Chapter 11 Kendall/Hunt Publishing Company 2 Historical Information 1953---James Watson and Francis Crick— discovered the configuration of the DNA molecule 1980---Ray White describes first polymorphic RFLP marker 1985---Alec Jeffreys isolated DNA markers and called them DNA fingerprints 1983---Kary Mullis developed PCR testing 1988—FBI starts DNA casework 1991—first STR research paper published 1994---DNA Identification Act initiates database/CODIS 1998—FBI launches NDIS/CODIS database Chapter 11 Kendall/Hunt Publishing Company 3 People of Historical Significance James Watson, Francis Crick, and Maurice Wilkins jointly received the Nobel Prize in 1962 for their determination of the structure of DNA. What is interesting about this fact is that Rosalind Franklin had as much to do with the discovery as the other three men with her work with X-ray crystallography. She died of cancer and could not be honored for her work. Kary Mullis received the 1993 Nobel Prize for his 1983 discovery of PCR, a way to quickly make copies of DNA in the lab. Chapter 11 Kendall/Hunt Publishing Company 4 General DNA Information Double helix—two coiled DNA strands Composed of nucleotides—units containing a sugar molecule (deoxyribose), a phosphate group and a nitrogen-containing base In humans, the order of these bases is 99.9% the same. Four bases Adenine Cytosine Guanine Thymine Bases always pair A to T and G to C Chapter 11 Kendall/Hunt Publishing Company 5 Where Is DNA Found? DNA is found in all nucleated body cells—white blood cells, semen, saliva, urine, hair root, teeth, bone, tissue Abundant in cheek cells Red blood cells have no nuclei, and therefore, no nuclear DNA DNA obtained from blood comes from white blood cells Genes are portions of DNA that code for specific proteins Chapter 11 Kendall/Hunt Publishing Company 6 Extracting DNA Break open the cell Release DNA from the proteins that package it in the cell Isolate the DNA from the extraction solution by precipitation Chapter 11 DNA Extraction Kendall/Hunt Publishing Company 7 DNA Typing DNA typing is a method in which DNA is characterized by length differences or sequence differences. Only 0.1 % of DNA (about 3 million bases) differs from one person to the next. Scientists use these regions to generate a DNA profile of an individual. Chapter 11 Kendall/Hunt Publishing Company 8 Non-Coding Regions 3 percent of the human DNA sequences code for proteins 97 percent is non-coding and is repetitive; repeating the same sequence over and over 50 percent of the human genome has interspersed repetitive sequences. These show great variety between people Chapter 11 Kendall/Hunt Publishing Company 9 Uses of DNA Profiling To identify potential suspects To exonerate individuals To identify source of biological evidence for reconstruction of crime To link or exclude individuals To identify crime and casualty victims To establish paternity To match organ donors Chapter 11 Kendall/Hunt Publishing Company 10 DNA TYPING RFLP—Restriction Fragment Length Polymorphism (fingerprinting) PCR—Polymerase Chain Reaction STR—Short Tandem Repeats Chapter 11 Kendall/Hunt Publishing Company 11 POLYMORPHISM MANY FORMS These are regions of DNA where individuals differ • They can differ by LENGTH • RFLP • STR • They can differ by SEQUENCE or ORDER • Dot blot • Sequencing • SNP Chapter 11 Kendall/Hunt Publishing Company 12 RFLP—Restriction Fragment Length Polymorphisms Restriction enzymes are used to cut DNA into smaller fragments that can then be separated and characterized for identification Isolate—separate DNA from the cell Cut—using restriction enzymes to make shorter base strands Sort—by size using electrophoresis Probe selected regions of DNA Analyze—the specific alleles for identification Chapter 11 Kendall/Hunt Publishing Company 13 RFLP The first DNA typing used for forensic work Advantage: Very discriminating Disadvantage: Requires a large sample Chapter 11 Kendall/Hunt Publishing Company 14 Electrophoresis Pipette the DNA. Chapter 11 Kendall/Hunt Publishing Company 15 Electrophoresis Load DNA into the gel wells. Chapter 11 Kendall/Hunt Publishing Company 16 Electrophoresis Run the gel. Observe and compare bands of DNA. Chapter 11 Kendall/Hunt Publishing Company 17 PCR—Polymerase Chain Reaction PCR is a technique used for making copies of a defined segment of a DNA molecule. This can be valuable when the amount of evidence is minimal. Millions of copies of DNA can be made from a single speck of blood. The PCR Song Chapter 11 Kendall/Hunt Publishing Company 18 The Brains Behind PCR Kary Mullis This link takes you to the Nobel Prize site which has a great deal of information about PCR, as well as an interview of Dr. Mullis. Chapter 11 PCR—Polymerase Chain Reaction Procedure Mix DNA, polymerase, nucleotides Heat to denature...separate strands Cool to anneal the primer...target area of interest (loci) Heat to extend...copy Repeat 30 times for about one billion copies! Each cycle takes about 2 minutes and doubles the number of copies Chapter 11 Kendall/Hunt Publishing Company 20 Test your PCR knowledge Play this game: PCR Mystery Game: The Eye of the Donkey More on PCR: See power of PCR multiplication Chapter 11 Kendall/Hunt Publishing Company 21 Advantages of PCR Tiny amounts of DNA may be tested Degrade DNA may be tested. Large numbers of copies of specific DNA sequences at different regions of DNA (loci) can be amplified simultaneously with multiplex PCR reactions. Commercial kits are now available for easy PCR reaction setup and amplification. Contaminant DNA, such as fungal and bacterial sources, will not amplify because human-specific primers are used. However, human contamination can be a problem. Chapter 11 Kendall/Hunt Publishing Company 22 Electrophoresis A technique used to separate DNA fragments. An electrical current is moved through a gel substance causing molecules to sort by size. The smaller, lighter molecules will move the furthest on the gel. The fragments can be visualized for characterization. Chapter 11 Kendall/Hunt Publishing Company 23 Short Tandem Repeats (STR) STR is a method of DNA typing. STR’s are locations (loci) on the chromosome that contain short sequences of 2 to 5 bases that repeat themselves in the DNA molecule. The advantages of this method are that it provides greater discrimination, requires less time, a smaller sample size, and the DNA is less susceptible to degradation. Chapter 11 Kendall/Hunt Publishing Company 24 Chapter 11 Kendall/Hunt Publishing Company 25 Short Tandem Repeats (STR) Procedure Extract the DNA Amplify the sample by means of PCR Separate by electrophoresis Examine the distance the STR migrates to determine the number of repeats Chapter 11 Kendall/Hunt Publishing Company 26 STR Gels Chapter 11 Kendall/Hunt Publishing Company 27 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. Chapter 11 Kendall/Hunt Publishing Company 28 Short Tandem Repeats (STR) Each person has two STR types at each locus, one inherited from each parent. By continuing the process with additional STRs from other genes, you can narrow down the probability of DNA belonging to only one probable person. Chapter 11 Kendall/Hunt Publishing Company 29 Profiler Plus Allelic Ladders VWA D3S1358 AMEL D8S1179 D5S818 FGA D21S11 D13S317 D18S51 D7S820 COfiler Allelic Ladders D3S1358 AMEL D16S539 TH01 TPOX CSF1PO D7S820 STR Example Determining Probability Chapter 11 Databases determine how often a particular allele on a loci appears in a given population. By increasing the number of alleles on different loci the probability of having two people with the exact combination becomes astronomical. Follow the product rule for independently inherited traits. Kendall/Hunt Publishing Company 34 DNA Interactive The website below has a STR animation demonstration. Click on human identification, profiling and then on the third circle called Today’s DNA Profiling to see the demonstration. http://www.dnai.org/d/index.html Chapter 11 Kendall/Hunt Publishing Company 35 Three Possible Outcomes Match—The DNA profile appears the same. Lab will determine the frequency. Exclusion—The genotype comparison shows profile 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. Chapter 11 Kendall/Hunt Publishing Company 36 Types of DNA Nuclear Mitochondrial found in the nucleus found in the cytoplasm constitutes 23 pair of is inherited only from chromosomes inherited mother from both parents each cell contains each cell contains only hundreds to thousands one nuclei of mitochondria can be found in skeletal remains, degrade, or low quantity samples Chapter 11 Kendall/Hunt Publishing Company 37 Mitochondrial DNA Analysis of mDNA is more: rigorous time consuming costly than nucleic testing of DNA mDNA is constructed in a circular or loop 37 genes are involved in mitochondrial energy generation Is used when nuclear DNA typing is not possible Chapter 11 Kendall/Hunt Publishing Company 38 FBI’s CODIS DNA Database Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1998 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Chapter 11 Kendall/Hunt Publishing Company 39 The Future Greater automation of the DNA typing process Use of SNP’s—single nucleotide polymorphism which measures a one nucleotide change or difference from one individual to another. More sites are needed to differentiate between individuals (30 to 50 SNPs to attain the frequencies of the 13 STR loci), but it can be done with robots and automation. Chapter 11 Kendall/Hunt Publishing Company 40 People in the News Sir Alec Jeffreys is credited with DNA profiling using RFLP. In September of 1984 after years of work, he saw his first series of blots on an X-ray. The technique was first used in forensics, when in 1985 he was asked by police to confirm the rape confession of 17 year old Richard Buckland, who was denying a rape of another young woman. he DNA from Buckland and the DNA taken from the victims eliminated him as a suspect. Jefferys then used samples from other suspects to later convict Colin Pitchfork whose DNA did match. Chapter 11 Kendall/Hunt Publishing Company 41 More about DNA For additional information about DNA and some famous cases, check out Court TV’s Crime Library at: www.crimelibrary.com/criminal_mind/forensics/dna/1.html Chapter 11 Kendall/Hunt Publishing Company 42