Chapter 11 DNA Analysis “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

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company DNA Analysis Students will be able to: Explain that DNA is a long molecule, tightly packed in the form of a chromosome containing genetic material. Isolate and extract DNA from cells. Describe the PCR process Describe STR testing and calculate probabilities of identity using STR. Kendall/Hunt Publishing Company

Historical Information James Watson and Francis Crick—1953 discovered the configuration of the DNA molecule Ray White—1980 describes first polymorphic RFLP marker Alec Jeffreys—1985 isolated DNA markers and called them DNA fingerprints Kary Mullis—1985 developed PCR testing 1988—FBI starts DNA casework 1991—first STR paper 1994—FBI launches CODIS database Kendall/Hunt Publishing Company

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. Find out more at Chemical Achievers: Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Where Is DNA Found? Genes are portions of DNA that code for specific proteins 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 Kendall/Hunt Publishing Company

General DNA Information Double helix—two coiled DNA strands Composed of nucleotides—units containing a sugar molecule (deoxyribose), 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 Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company How do we get the DNA out? EXTRACTION PROCEDURES: Release from cell Isolate and purify DNA EXTRACTION Kendall/Hunt Publishing Company

Why such a strange structure? It's a molecular Xerox! Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company We Are the World We are the World Kendall/Hunt Publishing Company

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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company DNA Interactive The website below has an STR animation demonstration. Click on human identification, profiling and then on the third circle called Today’s DNA Profiling to see the demonstration. Early DNA typing to Today's DNA typing PCR lab simulation Kendall/Hunt Publishing Company

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 Kendall/Hunt Publishing Company

Something to sing about... THE PCR SONG Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company The PCR Song by Scientists for Better PCR There was a time when to amplify DNA, You had to grow tons and tons of tiny cells. (Oooh) Then along came a guy named Dr. Kary Mullis, Said you can amplify in vitro just as well. Just mix your template with a buffer and some primers, Nucleotides and polymerases too. Denaturing, annealing, and extending, Well it’s amazing what heating and cooling and heating will do. [Chorus] PCR when you need to detect mutation (detect mutation) PCR when you need to recombine (recombine) PCR when you need to find out who the daddy is (who’s your daddy?) PCR when you need to solve a crime (solve a crime) [x2] Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company DNA Typing DNA typing is a method in which DNA is characterized by length or sequence differences. Only one-tenth of a single percent 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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

DNA TYPING “Fingerprinting” RFLP—Restriction Fragment Length Polymorphism PCR—Polymerase Chain Reaction STR—Short Tandem Repeats Kendall/Hunt Publishing Company

Two kinds of differences SEQUENCE DNA is a long molecule. DNA is a lung molecule. GTCAGTCAGGG GTCCGTCAGGG LENGTH DNA is a long long long long molecule. DNA is a long long molecule. CATCATCAT CATCATCATCATCAT Kendall/Hunt Publishing Company

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 Kendall/Hunt Publishing Company

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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

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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. Kendall/Hunt Publishing Company

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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Electrophoresis Pipette the DNA. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Electrophoresis Load DNA into the gel wells. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Electrophoresis Run the gel. Observe and compare bands of DNA. Kendall/Hunt Publishing Company

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Profiler Plus Allelic Ladders D3S1358 VWA FGA AMEL D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820

COfiler Allelic Ladders D3S1358 D16S539 AMEL TH01 TPOX CSF1PO D7S820

STR Example

Kendall/Hunt Publishing Company 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. Kendall/Hunt Publishing Company

Determining Probability Databases have been established that 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. Kendall/Hunt Publishing Company

Product Rule at its best! Testing only 4 loci, we get these results Locus 1: 5 (1/10) 12 (1/50) Locus 2: 10 (1/100) 20 (1/40) Locus 3: 6 (1/4) 8 (1/5) Locus 4: 4 (1/3) 9 (1/60) Multiply those together, and what do you get? Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 7.2 BILLION 1/7.200000000 1/7.2 billion! What is the population of the world? 7 billion And that is with only 4 loci. Most labs test at least 13! Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Types of DNA Nuclear found in the nucleus constitutes 23 pair of chromosomes inherited from both parents each cell contains only one nuclei Mitochondrial found in the cytoplasm is inherited only from mother each cell contains hundreds to thousands of mitochondria can be found in skeletal remains, degrade, or low quantity samples Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company FBI’s CODIS DNA Database Combined DNA Index System Used for linking serial crimes and unsolved cases with repeat offenders Launched October 1994 Links all 50 states Requires >4 RFLP markers and/or 13 core STR markers Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company E:\FORENSICS 1011\Forensic Lessons\FORENSIC SCIENCE CH 11 DNA 0809\codis.pdf Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company CODIS STATS UPDATE Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company PA Senate Bill 775 PA House Bill 713 Kendall/Hunt Publishing Company

Is it worth it? Ask the families of at least 10 victims. Consider the Grim Sleeper Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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. Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company 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 Kendall/Hunt Publishing Company

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 Kendall/Hunt Publishing Company

Kendall/Hunt Publishing Company Mullis Interview The website below has an interview of Nobel Prize winner, Dr. Kary Mullis, complete with some deep advice for students from one of the century’s greatest minds. http://nobelprize.org/mediaplayer/index.php?id=428 Kendall/Hunt Publishing Company