1. DNA Structure

2. What do these items have to do with one another?

3. Deoxyribonucleic Acid (DNA)
Forensic Science

4. 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
Video

5. Structure of DNA
double helix
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

6. 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.
DNA

7. 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.
DNA

8. 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

9. DNA at Work DNA 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

10. 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.
DNA

11. Intracellular DNA Replication
(Inside the cell)
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).

12. 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.

13. 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

14. 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
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15. PCR All the ingredients are placed into the thermal-cycler. PCR uses:
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.
PCR uses:
primer
DNA polymerase (taken from bacteria)
nitrogen bases (Adenine, Thymine, Cytosine and Guanine) to copy a segment of DNA.
Very little DNA is needed (0.2 μl)

16. Steps of PCR
Extract DNA
Denature
Anneal.
Extend.

17. 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

18. 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

19. 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

20. 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

22. 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
With these bands, we can compare suspect and crime scene DNA, or child and possible father, etc.

23. 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.

24. 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

25. 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

26. 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

27. 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

28. 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
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29. RFLP Analysis

30. Electrophoresis
A technique used to separate DNA fragments

31. 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

32. 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

33. Electrophoresis

34. 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

35. 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:
provides greater discrimination
requires less time
a smaller sample size,
DNA is less susceptible to degradation.

36. 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…

37. 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.

38. The CODIS loci analyzed by STR analysis
The CODIS loci analyzed by STR analysis. Notice they are spread over 14 chromosomes, and that two are on the X and Y chromosomes.

39. 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.

40. Combined DNA Information System (CODIS)
CODIS maintains a database of DNA profiles from
convicted offenders
unsolved crime scene evidence
profiles of missing persons

41. Possible Sources of DNA
Skin
Sweat
Blood
Mucus
Saliva
Tissue
Semen
Urine
Hair (root)
Ear Wax
Vaginal or rectal cells

42. 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

43. (end)

44. (stop)

45. Isolating DNA The first step to DNA sequencing is isolating DNA
Perform DNA Extraction Lab

46. DNA Sequencing: PCR

47. 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

48. (stop)
PCR Simulation

49. Extracellular DNA Replication
(Outside the cell)
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.

50. Steps of PCR
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.
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.
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 nucleotides/sec.

51. 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