ESPM 150/290Lecture 6: Forensic Genetic

Analysis24 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)

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

From Tissues to Cells to GenesFrom Tissues to Cells to Genes

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:

Deoxynucleotides (building blocks of DNA)

ACGTTGATCCTCATTGGA

CTGATCTTTAGGTCCAGC 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

Template DNA

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Primers

Newnucleo-tides

3 5

3

2

5 31

Taqpolymerase

(Thermus aquaticus)

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

Step 3: Sequence DNA » Most current applications use Sanger sequencing, which involves a modified

PCR reaction that includes chain-terminating, 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

dNTP

ddNTP

polymerase

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

agreed-upon 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

CCTATACCTAATCTTCGGAGCATGAGCGGGCATGGTAGGC...

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

Powersource

Gel

AnodeCathode

– +

+–

Agarose Gel Electrophoresis: (a.k.a. “running a gel”): Separating DNA by

size

Powersource

Principles:

1. Negative charge on DNA backbone causes migration in an electric current

2. Larger fragment = slower migration through gel matrix

3. DNA-binding compounds (e.g., EtBr) cause fluorescence in UV light

Shortermolecules

Mixture ofDNA

molecules of

differentsizes

Longermolecules

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)

Left Top: http://kingsley.stanford.edu

Bottom: http://www.paternity.be/information_EN.html

This photo shows EarlWashington just before his release in 2001,after 17 years in prison.

These and other STR data exonerated Washington and led Tinsley to plead guilty to the murder.

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STRmarker 1

STRmarker 2

STRmarker 3

17, 19

16, 18

17, 19

13, 16 12, 12

14, 15 11, 12

13, 16 12, 12

• Frequent addition or subtraction of the number of repeats means that individuals often be distinguished using STR loci

Analyzing microsatellite data

Analyzing microsatellite data

1. Raw data(dalla Martha et

al., 2007)

2. Data converted to genetic distances(dalla Martha et al., 2007)

Samples of unknown origin can be assigned to species or geographic regions based on their multilocus

genotypes