Criminalistics Name:_____________________________________________ Per:____ H.O.-DNA and DNA Fingerprinting DNA Structure and Function A basic functional and structural element of all living things is the cell. Sometimes the cell functions on its own, as in red blood cells, or in groups, such as in tissues and organs. In the nucleus of the cell are chromosomes that are inherited from both parents. Chromosomes are long-chain DNA (deoxyribonucleic acid) molecules that are tightly bound in a specific structure. If a single DNA strand were stretched out, it would reach about 5cm in length! DNA is a long-chain molecule made of four bases that are paired and held together with hydrogen bonds and a sugar-phosphate backbone. The bases that pair are adenine (A) with thymine (T) and guanine (G) with cytosine (C). The adenine and thymine are connected with two hydrogen bonds while the cytosine and guanine are connected with three hydrogen bonds. Each of these bases contains the element nitrogen; they are sometimes referred to as nitrogen bases. Each nitrogen base is connected to a sugar molecule (deoxyribose) and a phosphate group. These together make up what is called a nucleotide unit. PAIRED BASE (A-T or G-C) + sugar + phosphate = nucleotide unit The average human DNA molecule contains approximately 100 million of these nucleotide groups! In humans, the order of these nucleotide bases is 99.9% the same. The unique sequence of the other 0.1% makes each human one of a kind (except for identical twins, who have identical DNA). The sequence of these bases is a code for specific amino acids which combine to make specific proteins. The human body has approximately 35,000 genes, which are simply portions of the DNA that code the information required to make specific proteins. Genes can be as short as 1,000 base pairs or as long as several hundred thousand base pairs. One gene gives the information for one cell to produce one protein. These proteins then determine human traits and functions. Each gene has a specific code for a specific body function; they are the fundamental unit of heredity, determining traits from hair color, eye color, and facial features to certain diseases or disorders. A particular gene can be carried by more than one chromosome (EX: eye color). FORENSIC USES OF DNA Blood and bodily fluids are the most common evidence that forensic investigators use for testing of DNA. Blood is made up of red blood cells (carry oxygen throughout the body), plasma (the fluid that carries the cells), platelets (facilitate clotting), and white blood cells (defend the body against infection). Red blood cells lack nuclei containing DNA so it is the white blood cells that forensic scientists are interested in. A single drop of blood may contain anywhere from 7,000 to 25,000 white blood cells with the nuclei containing DNA inside. A small sample with only a few white blood cells is enough to extract DNA, and using the PCR (polymerase chain reaction) method, billions of copies can be made for testing. DNA “fingerprinting” is a common way to identify people by their unique genetic code. It is currently being used to identify the perpetrator in a crime, to identify fathers in paternity cases, and to identify unknown remains in mass disasters and other situations. DNA is in every nucleated cell of the human body and be extracted from blood, semen, urine, bone, hair follicles, and saliva. DNA fingerprinting can be used to: Identify potential suspects whose DNA may match evidence left at the crime scene(s) Clear persons wrongly accused of crimes Identify crime and catastrophe victims Establish paternity and other family relationships Match organ donors with recipients in transplant programs Samples collected from a crime scene are examined to determine whether the sample is appropriate for DNA analysis. If a sample is to be analyzed, it must be properly prepared. First, the DNA is removed from the object it is attached to (for example, clothing, weapon, skin, etc), then it is extracted from the cell. To isolate the DNA, the cellular components, such as fats, proteins, and carbohydrates, must be removed. Then enzymes are used to release the DNA from the chromosomal packaging. Once the DNA is extracted, it is ready for characterization. METHODS OF DNA FINGERPRINTING There are four main procedures involved in DNA fingerprinting: (1) isolation of the DNA to separate it from the cell, (2) cutting with a restriction enzyme to make shorter base strands, (3) sorting the segments by size using an electrophoresis procedure, and (4) analyzing the resulting print by identifying specific alleles Isolation and Cutting Techniques 1. RFLP (Restriction Fragment Length Polymorphism) At this time, a whole DNA molecule is too complex for scientists to characterize completely, and therefore it cannot be used as individual evidence. The best that forensic scientists can do is to characterize pieces (or fragments) of DNA and use statistics to determine the likelihood of another individual having the same fragments. To characterize DNA, the scientist must cut it into smaller pieces. This is done using restriction enzymes. A restriction enzyme will recognize a specific sequence of bases and cut the DNA molecule at a specific point. For example a restriction enzyme EcoRi will cut DNA whenever it finds the sequence “GAATTC”. It will cut between the G and A, as in: Other restriction enzymes cut at different sites: Enzyme Cutting Site Bam HI GGATCC between the G and G Hae III GGCC between the G and C Pst I CTGCAG between the A and G Bgl II AGATCT between the C and T Once the DNA is cut into different sized fragments, these fragments are separated through electrophoresis using a gel and voltage source. This procedure separates the fragments according to their sizes. The fragments are very close together and there are so many of them that it is difficult to make them visible. A probe (dye) is added that will adhere to specific fragments. By using a development technique the scientist can observe the new pattern, analyze an unknown sample from a crime scene, and compare it to the DNA of a suspect to see if it runs through the electrophoresis in the same manner. 2. PCR (Polymerase Chain Reaction) In many forensic cases there is very little evidence to work with. A technique called PCR offers the possibility for increased sensitivity in DNA fingerprinting. It can take a very small sample of DNA and make millions of copies by a relatively simple, quick method. PCR requires about 50 times less DNA than what is required for RFLP. Using the fact that base pairs are connected together with hydrogen bonds, which are rather weak, the strand is divided lengthwise and the new base pairs attach to new strands. Done repeatedly, this method can make millions of copies in a short time. In forensic applications, PCR has been able to identify perpetrators from as small a sample as saliva residue on a cigarette butt, a stamp, or the adhesive on an envelope. DNA is taken out of a small amount of blood, semen, or saliva in the same way as discussed earlier (by breaking down the cell wall and unwrapping the chromosome). The next step in PCR is to break down the DNA strands by heating. The heat separates the weak hydrogen bonds holding the base pairs together, leaving each DNA strand as two half- strands. The next step is to cool the mixture and add a primer, which is a short sequence of base pairs that will add to its complementary sequence on the DNA strand. The function of the primer is to begin the replication process. An enzyme called DNA polymerase is added along with a mixture of free nucleotide bases (A, T, G, and C), which then combine to their complementary bases on the free strand. This reaction works best at around 75 оC so the mixture is heated once again. Once the primer is in place, the polymerase can take over making the rest of the new chain. The two half-strands have now become four complete strands of DNA. After another cycle, there will be eight full strands, and so on. The three steps in PCR (separation, adding primer, and synthesis of new chain) only take about two minutes (mostly because of the heating and cooling). At the end of the cycle every strand of DNA is duplicated. It takes about 3 hours to make 1 million copies that can further be characterized. If the cycle was repeated 30 times, more than a billion copies could be produced! When DNA is so greatly amplified its typing or characterization can be simplified by methods that are not as complex as RFLP. One method is to add the DNA to a nylon strip that contains genetic markers, or alleles, that will bind to specific sequences of the DNA. These sequences can then be visualized and characterized. When several markers are used on several strips, the frequency of occurrence can be greatly reduced. 3. STR (Short Tandem Repeats) A new technology in the analysis of DNA is short tandem repeats (STR). This method is becoming more common than RFLP because it takes less time for the analysis, takes less of a sample size, and is more exclusionary (which means that it can eliminate more people as possible sources). STRs are locations on the chromosome that repeat a specific sequence of 2 to 5 base pairs. For the analysis, scientists identify multiple locations. A variable number of tandem repeats (VNTR) is also used, identifying repeats of 9 to 80 base pairs. Hundreds of SRT sites have been identified; they are located on almost every chromosome in the human genome. They can easily be amplified, using PCR, and characterized based on the alleles. Alleles are generally named by the number of repeats that they contain. For example, D7S280 is an STR found on human chromosome 7 that repeats the sequence GATA. The DNA sequence of the representative allele of this locus is shown below. Find the repeat sequence of GATA. How many repeats are shown on the DNA sequence below? Different alleles of this locus may have from 6 to15 tandem repeats of GATA. 1 AATTTTTGTA TTTTTTTTAG AGACGGGGTT TCACCATGTT GGTCAGGTG ACTATGGAGT 61 TATTTTAAGG TTAATATATA TAAGGGTAT GATAGAACAC TTGTCATAGT TTAGAACGAA 121 CTAACGATAG ATAGATAGAT AGATAGATAG ATAGATAGAT AGATAGATAG ATAGACAGAT 181 TGATAGTTTT TTTTTATCTC ACTAATAGT CTATAGTAAA CATTTAATTA CCAATATTTG 241 GTGCAATTCT GTCAATGAGG ATAAATGTGG AATCGTTATA ATTCTTAAGA ATATATATTC 310 CCTCTGAGTT TTTGATACCT CAGATTTTAA GGCC To identify individuals, forensic scientists scan 13 DNA regions that vary from person to person; they then use the data to create a DNA profile of the individual. There is an extremely small chance that another person has the same DNA profiles from a particular set of regions. D7S280 is one of the 13 core CODIS STR genetic loci. The probabilities of the STRs used can be multiplied together to narrow the field of suspects. The 13 standard CODIS STRs that the FBI uses to maintain their databank and their probability of identity are given in the following chart: STR African American American Caucasian D3S1358 0.097 0.080 VWA 0.074 0.068 FGA 0.036 0.041 TH01 0.114 0.080 TPOX 0.091 0.207 CFS1PO 0.079 0.128 D5S818 0.121 0.166 D13S317 0.139 0.081 D7S820 0.087 0.067 D8S1179 0.080 0.069 D21S11 0.042 0.041 D18S51 0.032 0.032 D16S539 0.076 0.091 If only one STR, D3S1358, were used, the likelihood that two African American individuals selected at random would be the same would be 1 in 10.3. Using the table above, it is calculated as follows: 1/X = 0.097, with X as the number of individuals in a sample. When you solve for X (X = 1/0.097) you get 10.3. Is this an acceptable probability to be certain the individual tested is guilty of a crime? What if 2 STRs were used? Try D3S1358 and FGA. You would multiply the probabilities of each event occurring and get: (0.097) * (0.036) = 0.0035. When you solve for X (X = 1/0.0035) = 285.7, or one in 285.7 people. The probability is getting better, but it’s still not good enough. Forensic scientists will use several of the STR sites to continue to narrow the possible field of suspects. If all 13 STRs are used to profile an individual, multiplying all the probabilities together can narrow the field (or frequency of occurrences) to one in billions. The FBI maintains a forensic index that has DNA profiles from crime scene evidence and an offender index with DNA profiles of individuals convicted of sex offenses and other violent crimes. All 50 states have become users and contributors to the indexes. Matches made among profiles in forensic science can link crime scenes together, possibly identifying repeat offenders. Based on a match, police in different jurisdictions can coordinate their investigations and share the leads they developed independently. Matches made between the forensic and offender indexes provide investigators with the identity of the perpetrator(s). After CODIS (Combined DNA Index System) identifies a potential match, the qualified DNA sample analysts in the laboratories contact each other to validate or refute the match. MITOCHONDRIAL DNA Another structure in the cell that contains DNA is the mitochondria. The mitochondria are considered the powerhouses of the cell, providing 90% of the energy a human needs to function. Each cell contains thousands of mitochondria, each containing several loops of DNA. Unlike nuclear DNA, which is found on the chromosomes and inherited from both the mother and father, mitochondrial DNA (mDNA) is inherited only from the mother. This makes individuals with the same maternal lineage indistinguishable if mitochondrial DNA is used for analysis. The techniques scientists use to characterize mDNA are significantly more sensitive than techniques for profiling nuclear DNA; however, analysis of mDNA is more costly and takes more time. An advantage of mDNA testing is that is can be done with small and degraded quantities of DNA. Currently, the FBI maintains one of the few labs that will do mDNA testing and they have strict limitations as to what types of cases they will accept.
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