ESPM 150/290
Lecture 6: Forensic Genetic Analysis
24 February, 2011
Guest Lecturer: Todd Osmundson, Ph.D.
Postdoctoral Researcher, Garbelotto Lab
The case of Colin Pitchfork
» Narborough, Leicestershire, England, 1983 and 1987:
2 brutal rapes/murders of 15-year-old girls unsolved.
Cases so closely matched that police strongly believe a
single suspect committed both.
» 1984, Leicester University: Professor Alec Jeffreys
develops techniques for DNA fingerprinting
» A 17-year-old suspect first denied involvement, but
under extensive questioning admitted to the second
but not the first murder
» Genetic comparison of crime scene and suspect’s
blood samples showed he was not responsible for
either murder. Thus, Richard John Buckland was the
first person exonerated of a crime by DNA evidence.
The case of Colin Pitchfork
» Police subsequently took blood samples from every
13-30-year-old man in 3 local villages
» A local bakery owner overheard a conversation where
one man bragged about paying someone else to
provide a sample on his behalf, reported him to
police, and man was apprehended
» DNA evidence implicated the man, Colin Pitchfork,
in the crime -- the first person to be convicted based
on genetic fingerprinting
How can we assess the relatedness
of individuals?
Molecular genetic approaches to assessing
relatedness
» What is phenotype?
» Physical appearance or other physically observable
manifiestation (biochemical, physiological, behavioral,
ecological) of the genotype
» Product of genotype-environment interaction
» The unit upon which natural selection acts; can lead to
phenotypic convergence
» Genotype: The genetic composition (i.e., DNA sequence) of
an organism. The unit of inheritance.
» What can we gain from observing the genotype directly?
» Direct observation of genetic relatedness
» Characters may be more objective to assess (not always true,
however)
From Tissues to Cells to Genes
Sources: (clockwise from upper left: http://www.healthinmotion.net/HIM/HTM/LS.html;
http://www.alzheimers.org/rmedia/IMAGES/LOW/Dna_low.jpg; http://radiographics.rsna.org
DNA Structure
• DNA is the genetic information-carrying
molecule in a cell
• 4 building blocks (bases): Adenine,
Cytosine, Guanine, and Thymine
A and T bind together
G and C bind together
• 2 strands arranged in a double helix
• The sequence of a piece of DNA is the
order of its bases, depicted as a string of
letters (e.g., TGCATTACTACGTG)
• Because of the predictable pattern of
complementary binding (A +T, G +
C), if we determine the sequence of one
strand, we automatically know the
sequence of the other strand
A Generalized DNA Workflow
Step 1: Extract DNA from cells
» Physically disrupt tissue to expose cells
and break cell walls
» Add detergent to break down cell
membranes
» Use chemical methods to separate DNA
from proteins, cell wall debris, and other
cellular components
Photo: Gero Steinberg, University of Exeter
Step 2: Amplify DNA
»
»
Large amounts of DNA are needed for analysis. The Polymerase Chain
Reaction (PCR) allows the selective amplification (i.e., making many copies of a
particular DNA region of choice).
Takes advantage of the complementary binding of DNA and the DNA-copying
action of primers and DNA polymerase enzymes (i.e., normal cellular
mechanism for copying DNA)
Ingredients:
Template DNA
Deoxynucleotides (building blocks of DNA)
Primers (starting points for DNA synthesis); DNA
(not RNA as in normal DNA replication)
Thermostable DNA polymerase (executes DNA synthesis), plus
buffer and MgCl2 to make it work
CTGATCTTTAGGTCCAGC
ACGTTGATCCTCATTGGA
1 Denaturation
5
3
3
5
2 Annealing
Cycle 1
yields
2
molecules
Primers
3 Extension
Taq
polymerase
(Thermus aquaticus)
New
nucleotides
Cycle 2
yields
4
molecules
Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Step 3: Sequence DNA
» Most current applications use Sanger sequencing, which
involves a modified PCR reaction that includes chainterminating, fluorescently-labeled versions of the bases
(each type of base has a different color fluorescence signal)
» Different points of incorporation of chain-terminating bases
yields many DNA copies of varying lengths. An automated
sequencing machine reads both the length and the
fluorescence signal for each fragment, and puts this
information together to produce a chromatogram. The
machine also assembles the information into a sequence
text file.
Step 3: Sequence DNA
Step 3: Sequence DNA
Step 3: Sequence DNA
Step 3: Sequence DNA
polymerase
ddNTP
dNTP
Step 3: Sequence DNA
Step 3: Sequence DNA
Step 3: Sequence DNA
Step 4: Analyze
Sequence chromatograph
Step 4: Analyze
»
Example: Sequence comparisons. The figure shows an example of a sequence
alignment. Each row represents the sequence data for one sample. Each vertical
column represents one identical position in the sequence between samples.
Within a column, differences between rows represent evolutionary changes
(mutations, insertions/deletions)
Step 4: Analyze
»
Example: querying the GenBank database to determine the identity or putative
function of an unknown DNA sequence by comparing it to the sequence of
known genes.
Step 4: Analyze
»
Example: phylogenetic tree building. Group organisms by analyzing changes
between DNA sequences using a selected criterion (e.g., optimizing the
arrangement of relationships so that it contains the least number of total
changes, or so that it is the most probable given a model of how sequences are
believed to change over time).
Figure: Greg Mueller, The Field Museum
DNA Barcoding
» Identification of species by sequencing an agreedupon gene (cytochrome oxidase 1 for most animals;
rDNA internal transcribed spacer for fungi)
» Assuming that each species differs in the sequence of
this gene (and that the gene sequence is constant
within a species), each species will have a unique
genetic code, analogous to the supermarket UPC
code.
DNA Barcoding Workflow
CCTATAC
CTAATCT
TCGGAG
CATGAGC
GGGCAT
GGTAGG
C...
Multilocus Genotyping
» What if we want to compare 2 closely-related
individuals?
» Will their genotypes be similar?
» Do we have to look at more, or fewer, data when
comparing closely-related (compared to distantly-related)
individuals?
Molecular Markers
» Inherited genotypic
Agarose Gel Electrophoresis: (a.k.a. “running a
gel”): Separating DNA by size
Cathode
Mixture of
DNA
Principles:
molecules
of
1. Negative charge on DNA
different
backbone causes
sizes
migration in an electric
–
Power
source
Anode
+
Gel
current
2. Larger fragment = slower
migration through gel
matrix
3. DNA-binding compounds
(e.g., EtBr) cause
fluorescence in UV light
Power
source
–
+
Longer
molecules
Shorter
molecules
RESULTS
+
Microsatellites (Simple Tandem Repeats)
• Regions where a small motif of nucleotides (CA in the
example below) is repeated multiple times. Can occur in
coding or noncoding regions of the genome.
• DNA polymerase has a difficult time faithfully
reproducing STRs, therefore, addition or subtraction of the
number of repeats happens relatively frequently. The
number of repeats, like other aspects of DNA, is heritable.
Microsatellites (Simple Tandem Repeats)
• Frequent addition or subtraction of the number of repeats
means that individuals often be distinguished using STR loci
This photo shows Earl
Washington just before
his release in 2001,
after 17 years in prison.
Source of
STR
sample
marker 1
Semen on victim17, 19
Earl Washington 16, 18
STR
marker 2
13, 16
STR
marker 3
12, 12
14, 15
11, 12
13, 16
12, 12
Kenneth Tinsley 17, 19
These and other STR data exonerated Washington
and led Tinsley to plead guilty to the murder.
Left Top: http://kingsley.stanford.edu
Bottom:
http://www.paternity.be/information_EN.html
Analyzing microsatellite data
Analyzing microsatellite data
2. Data converted to genetic distances
(dalla Martha et al., 2007)
1. Raw data
(dalla Martha et al.,
2007)
Samples of unknown origin can be assigned
to species or geographic regions based on
their multilocus genotypes