Science

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Forensic DNA Analysis Part 1: The science behind DNA profiling Part 2: The case of the Romanovs Part 3: Analyzing the certainty of DNA profile comparisons Part 4: Uncertainties in DNA profiling

and privacy issues

Part 1: The science behind DNA Profiling

*Frumkin et al., Forensic Science International: Genetics, 2010, 4(2) pp. 95-103

*

*

*

‡‡Bright and Petricevic, Forensic Science International, 2004, 145, pp.7-12.

‡ ‡

‡ ‡

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DNA is one of many forms of physical evidence

•  Fingerprints •  Blood •  Guns •  Knives •  Bullets •  Wrecked cars •  Human bones •  Bruises •  Clothing •  Computers •  Papers

Image: Simon Howden / FreeDigitalPhotos.net

Collection of DNA evidence •  Trace amounts of DNA can be

recovered from –  Hair follicles –  Scrapings of dead skin (e.g

from under someone’s fingernails)

–  Saliva –  Semen –  Blood –  Bone fragments buried for

hundreds of years (and including teeth)

•  Collection of DNA evidence (from a crime scene for example) must be done carefully to avoid: –  Contamination –  Degradation –  The mixing up of samples

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DNA is small

•  A DNA molecule is ~20 nm (1 nm = 1.0 x 10-9 meters) wide

•  Found in the nucleus of a cell •  Human cells are ~5-30 µm (1

µm = 1.0 x 10-6 m) wide, too small to be visible to the naked eye

•  There is about 6 pg DNA in typical human cell (1 pg = 1.0 x 10-12 g)

Illustration of a DNA double helix

Illustration of an animal cell

~20 nm

~30 µm

Why is DNA good evidence? •  Every person’s DNA sequence is

unique •  The “sequence” of the DNA refers

to how the DNA basepairs are arranged, like letters in a word or words in a sentence

•  Homozygotic (ie “Identical”) twins have DNA sequences that are the most similar

•  Blood relatives have DNA sequences that are more similar than unrelated individuals

•  Unrelated individuals have DNA that is VERY different from each other, making DNA more helpful in crime scene investigation than less “individual-specific” evidence such as hair color or clothing fibers

Cartoon drawing of DNA double helix

DNA basepairs

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How Mendelian Inheritance Influences DNA sequence

•  You have 2 copies of every chromosome

•  You inherit one copy (and its sequence) from each of your parents

•  Which of each of your parents’ two copies you inherit is random

•  So for a given chromosome, if Mom is “chocolate/ chocolate” and Dad is “vanilla/strawberry” then you can be either “chocolate/vanilla” or “chocolate/strawberry”

Mom Dad

Ch1 Ch1

Ch1 Ch1

You (possibility #1)

You (possibility #2)

Or

How Mendelian Inheritance Influences DNA sequence

•  The chromosomes segregate (separate) independently with respect to each other during cell division

•  This “independent segregation” phenomenon greatly increases the number of possible combinations

•  The many possible combinations is the main reason why siblings look different than each other (unless they are identical twins) even though they have the same parents.

Mom Dad

You (possibility #1)

You (possibility #2)

Ch1 Ch2 Ch1 Ch2

Ch1 Ch2 Ch1 Ch2

You (possibility #3)

You (possibility #4)

Ch1 Ch2 Ch1 Ch2

…etc

… etc

Or Or Or

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Methods of Analyzing Differences in DNA Sequences

•  An older method is called “RFLP” analysis (Restriction Fragment Length Polymorphism)

•  RFLP analysis makes use of pieces of DNA that are different sizes between individuals and uses the differences to distinguish people

•  A later modification of this was called “VNTR” analysis (Variable Number Tandem Repeat) and which was in use around the time of the OJ Simpson trial in 1995

National Institutes of Health website

The red bands represent DNA molecules; they appear at different vertical positions on the grey “Southern

blot” because they are differently sized.

Large

Medium

Small

Methods of Analyzing Differences in DNA Sequences

•  These days, analysis is usually done by “STR” analysis (Short Tandem Repeat)

•  This method also uses differences in DNA size, but the differences are smaller (a few DNA base pairs instead of thousands)

•  STR analysis requires a much smaller sample size (~1 ng) and it will work on old or degraded samples making it more useful in crime scene investigation

•  Analyzing more sites in one comparison increases the certainty of the analysis.

•  The FBI uses 13 STR sites (ie the 13 “CODIS loci”).

http://www.cstl.nist.gov/biotech/strbase/fbicore.htm

The 13 loci used by the FBI. “CODIS” is the “Combined DNA Index System”…the specific group loci (shown above) usually used by law enforcement.

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Separate strands

(denature)

Add primers (anneal)

Make copies (extend primers)

Repeat Cycle, Copying DNA Exponentially

Starting DNA Template

5’

5’

3’

3’

5’

5’

5’ 3’ 3’

3’ 3’ 5’ Forward primer

Reverse primer

Figure 4.2, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

STR Analysis

•  Portions of the trace DNA sample are copied many times using a laboratory technique called the “Polymerase Chain Reaction” (P CR)

STR Analysis

*Frumkin et al., Forensic Science International: Genetics, 2010, 4(2) pp. 95-103

*

6 8

** From Figure 6.10, J.M. Butler (2005) Forensic DNA Typing, 2nd Edition © 2005 Elsevier Academic Press

6

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•  Following PCR, the resulting DNA samples are abundant enough to have their sequence read by a DNA analyzer

•  The readings are peaks on an “electropherogram” as shown to the right

•  The peaks represent DNAs of different sizes that were separated

•  Peak sizes are calibrated against a set of “size markers” (i.e. DNA molecules of known size)

•  DNA from different people will exhibit differences in the numbers of copies of the tandem repeat regions.

•  The differing numbers of copies create size differences in the peaks

•  Even DNA from the two copies of the chromosomes in one person will give different peaks if the donor individual is heterozygous.

One chromosome (vanilla)

The other copy of the same chromosome

(strawberry)

**

**

The DNA repeat region

stutter peak

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Allele •  Any of the possible forms in which a gene for a

specific trait can occur. In almost all animal cells, two alleles for each gene are inherited, one from each parent. Paired alleles (one on each of two paired chromosomes) that are the same are called homozygous, and those that are different are called heterozygous. In heterozygous pairings, one allele is usually dominant, and the other recessive. Complex traits such as height and longevity are usually caused by the interactions of numerous pairs of alleles, while simple traits such as eye color may be caused by just one pair.

An Example of STR Analysis: Amelogenin DNA is Used to Determine Sex

•  Amelogenin is a protein required for the formation of tooth enamel

•  Both men and women have amelogenin

•  DNA coding for amelogenin is located on the sex chromosomes

•  A woman’s sex chromosomes are “XX”

•  A man’s sex chromosomes are “XY”

Electron micrograph of an “amelogenin gel matrix”

Fincham, A.G., Moradian-Oldak, J. and Simmer, J.P., Journal of Structural Biology, 1999, 126, pp. 270-299.

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Sequencing of Amelogenin DNA •  DNA corresponding to amelogenin

is slightly longer on the Y- chromosome than on the X

•  STR analysis of DNA from a male will show two separate signals (one signal each deriving from the X and Y chromosomes, respectively)

•  DNA from a woman shows only the shorter X signal since both X chromosomes report the same result X ≈ 103 DNA base pairs

Y ≈ 109 DNA base pairs

What results look like for a male:

Frumkin et al., Forensic Science International: Genetics, 2010, 4(2) pp. 95-103

Another example of how differences in DNA sizes can be used in DNA profiling: Paternity testing

•  This one is the older “VNTR” analysis

•  For every VNTR locus that can be analyzed in the laboratory, you will bear one of the two signals each of your parents do

•  You will not bear any VNTRs not borne by at least one of your parents

Morling, N. and Hansen, H., Int. J. Leg. Med., 1993, 105, pp. 189-196.

La dd

er

La dd

er

Mo th

er

Mo th

er

Ch ild

Ch ild

Ex clu

de d

m an

Ex clu

de d

m an

No n-

ex clu

de d

m an

No

n- ex

clu de

d m

an

Case 1 Case 2

..the so-called “obligate paternal allele”

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Summary of Part 1 of DNA Profiling

•  DNA evidence is just one type of evidence •  DNA can be valuable evidence because:

–  each person’s DNA is unique –  testing requires only a small DNA sample –  the sample can be old and somewhat degraded

•  STR analysis is currently the most commonly used method of DNA testing and relies on small variations in lengths of DNA that vary between individuals

•  Individuals that are blood relatives have DNA that is more similar than unrelated people and our detailed knowledge of this enables DNA tests with different purposes (i.e. crime scene investigations vs paternity testing)

Testing for blood at a crime scene •  CSI’s normally try to detect,

photograph and sample blood stains directly

•  When blood is too hard to detect directly, trace blood can be detected by spraying with luminol and an oxidizer (e.g. peroxide…. H2O2)

•  Drawback is that the luminol/ oxidizer mix will also react with other things that aren’t blood (ferricyanide, horseradish, plant roots and some bleaches, for example)

•  Another drawback is that contaminating the blood sample with luminol spray can limit or prevent other tests being run on the blood

Photo by” David Muelheims,”

Germany”

I.A.B.P.A. News March, 2007

500 mL of horse blood poured on the ground and monitored for 16 months in an experiment to determine how long “post- deposition” a bloodstain exposed to the elements would be detectable with luminol

Chemical structure of luminol

C

C C

C

C C

C N

N C

NH2 O

O

H2O2 O2 H2O Fe from hemoglobin

in blood as catalyst

Reaction between oxygen and luminol gives off blue light

H

H H

H

H

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Testing for blood at a crime scene •  The luminol is deprotonated by the base (OH-)

in mixture and becomes the dianion •  When located in near physical proximity (ie on

a blood stain), a catalyst such as the iron in hemoglobin from blood, disproportionates peroxide into molecular oxygen and water

•  Anything that can catalyze the formation of oxygen from peroxide will be a source of background (eg. Horseradish peroxidase in horseradish, as is commonly used in chemiluminescence detection of Western blots).

•  The O2 evolved by the disproportionation of hydrogen peroxide reacts with the dianionic form of the luminol to give a diphthalate in a triplet excited state

•  The triplet excited state undergoes an intersystem crossing to the singlet excited state which then gives off a blue photon when returning to the ground state

•  This mechanism which involves intersystem crossing technically makes the luminol glow a “phosphorescence” event because intersystem crossing is a “forbidden transition”. That it is a phosphorescence event is one of the reasons why the glow lasts a long time and is visible for 30 sec or so in the context of crime scene investigation.

H2O2 O2 H2O Fe from hemoglobin

in blood as catalyst

Wikipedia.com

Assignment

•  Read Article: “Establishing the identity of Anna Anderson Manahan”

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