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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