Post on 29-Dec-2015
DNA Structure
https://www.youtube.com/watch?v=qy8dk5iS1f0
What do these items have to do with one another?
Deoxyribonucleic Acid (DNA)Forensic Science
Introduction to DNA• Like fingerprints, DNA is unique to each
individual individual evidence!• DNA can definitively link a suspect to a victim or
crime scene.• Chromosomes are threadlike structures composed
of DNA• The primary unit of heredity is called a gene
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Video
Structure of DNA• DNA (deoxyribonucleic acid) is polymer of
repeating units called nucleotides– A nucleotide consists of
• Sugar-phosphate backbone– Deoxyribose sugar– Phosphate
• Nitrogenous base – adenine, guanine, cytosine, thymine
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double helix
The Bases• Four types of bases used
in DNA: adenine (A), guanine (G), cytosine (C), and thymine (T).
• Complementary base pairing occurs between A and T, and the base C with G in double stranded DNA.
The Bases
• DNA’s structure is a double-stranded helix as discovered by Rosalind Franklin, Crick and Watson.
• Chargaff discovered that A always pairs with T and C always pairs with G.
Structure of DNA• The long strands of DNA are coiled upon
themselves into chromosomes– When paired, chromosomes resemble the letter X
• Humans have 23 pairs of chromosomes, including 2 sex chromosomes– Females are XX– Males are XY
DNA at Work• DNA codes for proteins
• Proteins are formed by amino acids (monomers) in a long chain.
• The sequence of amino acids determines the shape and function of the protein.
DNA at Work• A codon (three nucleotides)
codes for a particular amino acid.
G-A-G codes for the amino acid glutamine, while C-G-T codes for alanine.
Intracellular DNA Replication• DNA duplicates itself in
the nucleus prior to cell division.
• DNA replication begins with the unwinding of the DNA strands of the double helix.
• Many enzymes are involved in unwinding the DNA (helicases), and assembling the new DNA strands (polymerases).
(Inside the cell)
Extracellular DNA Replication (Outside the cell)
Why would a forensic scientist want to replicate DNA?
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.
Extracellular Replication - Polymerase Chain Reaction (PCR)
• A technique for making many copies of a specific piece of DNA to be analyzed forensically– Can amplify very minute quantities of DNA millions
of times!
• This method works by cycling through different temperatures
• A device called a thermocycler controls the temperatures, allowing for fast and accurate copying of DNA
PCR
• The outcome is a doubling of the number of DNA strands.
• Heating, cooling, and strand rebuilding is repeated typically 25 to 30 times, yielding more than one million copies of the original DNA molecule.
• Each cycle takes less than two minutes from start to finish
Video - https://www.youtube.com/watch?v=0HCWmD7Mv8U
PCR• PCR uses:
o primero DNA polymerase
(taken from bacteria)o nitrogen bases
(Adenine, Thymine, Cytosine and Guanine) to copy a segment of DNA.
• Very little DNA is needed (0.2 μl)
• All the ingredients are placed into the thermal-cycler.
• Thermal-cycler heats the DNA sequence to unravel the strand.
• The A, T, C and G will pair with the sample in the correct order, the copy will be released when the cycle cools.
Steps of PCR
1. Extract DNA
2. Denature
3. Anneal.
4. Extend.
Polymerase Chain Reaction (PCR)The steps of PCR
• Denaturation: – Extracted and purified DNA is heated to “unzip”
(separate) the double helix– This is done at high temperature, about 94°C
Polymerase Chain Reaction (PCR)The steps of PCR
• Annealing: – Short template pieces called “primers” bind with
the separated strands for new DNA to build upon.– This occurs at ~65°C
Polymerase Chain Reaction (PCR)The steps of PCR
• Extend/Elongation:– DNA Polymerase adds free nucleotides from the
surrounding solution onto the template primers– In this way, new strands are built out of the original
2 separated stands– This happens at 72°C
new DNA strands
Polymerase Chain Reaction (PCR)• Each step only requires a few minutes• The thermocycler machine cycles through
these temperatures for several hours• Each cycle doubles the number of copied
DNA strands
PCR is specific to your “region of interest.” Different
primers will selectively amplify different genes
DNA Typing• DNA typing (a.k.a. DNA Fingerprinting) was
developed by British geneticist Sir Alec Jeffreys in 1984.
• This technique converts DNA into readable bands on a gel
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With these bands, we can compare suspect and crime scene DNA, or child and possible father, etc.
DNA Typing
DNA typing is a method in which DNA is converted into a series of bands that distinguishes each individual.
Only one-tenth of a single percent of DNA (about three million bases) differ from one person to the next. Scientists use these regions to generate a DNA profile of an individual.
Uses of DNA Profiling
To identify potential suspects
To identify crime and casualty victims
To establish paternity
To exonerate individuals
To match organ donors
jyokum 2013
DNA Typing
• Portions of the DNA molecule contain sequences of bases that are repeated numerous times, known as tandem repeats.
• Tandem repeats – region of a chromosome that contains multiple copies of a DNA sequence that repeats
• To a forensic scientist, these tandem repeats distinguish one individual from another through DNA-typing.
DNA Typing
• Tandem repeats are found throughout our genome between the coding areas of DNA.
• What is important to understand is that all humans have the same repeat regions, but there is tremendous variation in the number of repeats and each person has a unique number in each region of their genome.
DNA Typing“Fingerprinting” 1985—Alec Jeffreys
Forensic scientists aimed efforts at characterizing the following repeat segments:
• RFLP – restriction fragment length polymorphism
• STR – short tandem repeats
RFLP—Restriction Fragment Length Polymorphisms
Restriction enzymes are used to cut DNA into smaller fragments that can then be separated and characterized for identification.
1) Extract—separate DNA from the cell
2) Cut—use of restriction enzymes to make shorter base strands
3) Sort—by size using electrophoresis (DNA separation technique)
4) Analyze—the specific alleles for identification
Video - https://www.youtube.com/watch?v=jMLIaxxY6-8
RFLP Analysis
Electrophoresis
• A technique used to separate DNA fragments
Electrophoresis
• In the lab, DNA molecules are cut by restriction enzymes into fragments of various sizes. Restriction enzymes cut at specific sequences throughout the DNA.
• The resulting fragments are forced to move along a gel-coated plate under the influence of an electrical potential
Electrophoresis
• DNA samples are injected into a gel
• An electrical current is moved through a gel
• DNA molecules moves because it is negatively charged
• DNA molecules are sorted by size.
• The smaller, lighter molecules will move the farthest on the gel.
jyokum 2013
Electrophoresis
https://www.youtube.com/watch?v=lgmq_HsuZIU
Electrophoresis
• After the fragments have “migrated” across the gel, the gel can be stained to show the “bands” or fragments easily
• Then comparisons can be made– Example: crime scene
sample to suspect
Short Tandem Repeats (STR)
• STR is another method of DNA typing.
• STRs are locations (loci) on the chromosome that contain short sequences of two to five bases that repeat themselves in the DNA molecule.
• The advantages of this method over RFLP are:
1. provides greater discrimination
2. requires less time
3. a smaller sample size,
4. DNA is less susceptible to degradation.
Short Tandem Repeats (STRs)
• A common method of DNA typing
• STR’s are less susceptible to degradation and can be recovered from bodies or stains that have been subject to extreme decomposition
• With the technology of PCR one can extract and amplify a combination of different STR’s. More on this later…
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STRs
• In forensic laboratories, there are thirteen STR loci that are typically used to create a genetic fingerprint of an individual.
• Although controversial, the profile is kept in DNA databases. In the United States, the genetic fingerprints are kept in various Combined DNA Index Systems (CODIS) databases ranging from the smaller, local levels to the national level.
• The CODIS loci analyzed by STR analysis. Notice they are spread over 14 chromosomes, and that two are on the X and Y chromosomes.
CODIS
• The Combined DNA Index System is maintained by the Federal Bureau of Investigation. As of June 2012, CODIS maintained over 9.7 million offender profiles, 1.1 million arrestee profiles and 436,000 forensic profiles. Profiles maintained in CODIS are compiled from both suspects and evidence, and therefore are used to help solve criminal cases. Also as of June 2012, CODIS has produced over 182,200 "hits," assisting in more than 174,600 investigations.
• Profiles of missing persons are also maintained in CODIS. The true power of STR analysis is in its statistical power of discrimination. Because the 13 loci are independently assorted, the laws of probabilities can be applied. This means that if someone has the genotype of ABC at three independent loci, then the probability of having that specific genotype is the probability of having type A times the probability of having type B times the probability of having type C. This has resulted in the ability to generate match probabilities of 1 in a quintillion.
Combined DNA Information System(CODIS)
• CODIS maintains a database of DNA profiles from
–convicted offenders –unsolved crime scene
evidence–profiles of missing
persons
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Possible Sources of DNA
• Skin• Sweat• Blood• Mucus• Saliva• Tissue• Semen• Urine• Hair (root)• Ear Wax• Vaginal or rectal cells
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Collecting and Packaging Biological Evidence• Photograph evidence first• Wear gloves at all times• Package each stained article separately
in paper or a well-ventilated box (to avoid bacterial or fungal growth)
• Remove dried blood using a sterile swab moistened with distilled water
• Store biological evidence in the refrigerator or a cool location until it is delivered to the lab
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Isolating DNA
• The first step to DNA sequencing is isolating DNA
• Perform DNA Extraction Lab
DNA Sequencing: PCR
Polymerase Chain Reaction (PCR)
• A technique for making many copies of a specific piece of DNA to be analyzed forensically– Can amplify very minute quantities of DNA millions
of times!
• This method works by cycling through different temperatures
• A device called a thermocycler controls the temperatures, allowing for fast and accurate copying of DNA
(stop)
PCR Simulation
Extracellular DNA Replication
• Polymerase chain reaction (PCR) is a technique for replicating a small DNA sample found at a crime scene.
• The ability to multiply small bits of DNA means that a single sample is no longer a limitation in analyzing crime scene DNA.
(Outside the cell)
Steps of PCR1. Denature. The first step requires a high temperature to
denature and separate the double stranded DNA. This is done by heating the sample to 92-94oC.
2. Anneal. The second step requires lowering the temperature to allow annealing (binding) of the primers to the single stranded DNA. The optimal annealing temperature is 45-55oC.
3. Extend. The third step requires DNA synthesis by DNA polymerase. The optimal temperature is about 75-80oC. The rate of primer extension by polymerase is about 50-100 nucleotides/sec.
Resources• Saferstein, Richard. Forensic Science: An Introduction. New
Jersey: Pearson Prentice Hall, 2008• Saferstein, Richard. Forensic Science: An Introduction. 2nd
ed. New Jersey: Pearson Prentice Hall, 2011• Saferstein, Richard. Criminalistics: An Introduction to
Forensic Science. 8th ed. Upper Saddle River, NJ; Pearson Prentice Hall, 2004
• http://law2.umkc.edu/faculty/projects/ftrials/clinton/lewinskydress.html
• http://www.trutv.com/library/crime/notorious_murders/famous/simpson/index_1.html
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