0. 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

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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
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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.
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3. 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
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4. 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:
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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
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6. 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
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How do we get the DNA out?
EXTRACTION PROCEDURES:
Release from cell
Isolate and purify
DNA EXTRACTION
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8. Why such a strange structure?
It's a molecular Xerox!
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We Are the World
We are the World
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10. 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.
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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.
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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
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13. 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
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14. Something to sing about...
THE PCR SONG
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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]
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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.
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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
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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
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19. DNA TYPING “Fingerprinting”
RFLP—Restriction Fragment Length Polymorphism
PCR—Polymerase Chain Reaction
STR—Short Tandem Repeats
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20. 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
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21. 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
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22. 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.
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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.
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25. 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.
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26. 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.
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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.
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Electrophoresis
Pipette the DNA.
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Electrophoresis
Load DNA into the gel wells.
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Electrophoresis
Run the gel.
Observe and compare bands of DNA.
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32. Profiler Plus Allelic Ladders
D3S1358
VWA
FGA
AMEL
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820

33. COfiler Allelic Ladders
D3S1358
D16S539
AMEL
TH01
TPOX
CSF1PO
D7S820

34. STR Example

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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.
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37. 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.
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38. Product Rule at its best!
Testing only 4 loci, we get these results
Locus 1: (1/10) (1/50)
Locus 2: (1/100) (1/40)
Locus 3: (1/4) (1/5)
Locus 4: (1/3) (1/60)
Multiply those together, and what do you get?
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7.2 BILLION 1/
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!
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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
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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
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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
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E:\FORENSICS 1011\Forensic Lessons\FORENSIC SCIENCE CH 11 DNA 0809\codis.pdf
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CODIS STATS UPDATE
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PA Senate Bill 775
PA House Bill 713
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46. Is it worth it? Ask the families of at least 10 victims.
Consider the Grim Sleeper
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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.
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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.
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More about DNA
For additional information about DNA and some famous cases, check out Court TV’s Crime Library at:
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50. 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
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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.
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