Forensic DNA Analysis

SFS3f. Compare short tandem repeat patterns (STR) and relate to identifying the DNA of an individual.

SFS3g. Explain the use of the DNA database for DNA profiling.

EQ

What is DNA? How is DNA evidence analyzed by

Criminalist & how has it become useful to criminal investigations over the past quarter century?

What is DNA?

DNA “fingerprinting” is a common way to ID people by their unique genetic code.

DNA is a double stranded molecule located in the nucleus of a cell; Every cell in an organism contains the same DNA.

Characteristics of DNA varies btwn individuals w/in a species & btwn species.

What is DNA? DNA- deoxyribonucleic

acid. Long-chain molecule

made up of four bases that pair together & held together by hydrogen bonds.

A=T; G=C In humans- order of

bases are 99.9% the same.

What is DNA?

DNA is contained in chromosomes

Chromosomes contain DNA &

associated proteins (which allow DNA to condense/wind/coil up tightly)

What is DNA?

Genes are sequences of DNA nucleotides

Genes are located on chromosomes

Alleles: different types of the same gene (dominant, recessive, co-dominant, etc).

Forensic DNA analysis seeksnucleotide variation in DNA

1. Point mutations from errors in DNA

replication – RFLP (Restriction Fragment

Length Polymorphism) analysis 2. Variable number tandem

repeats (VNTRs) – STR (Short tandem repeat)

analysis

Why is there nucleotide variation between the DNA of individuals of the same species?

All the DNA nucleotides in a cell must be copied (replicated) prior to cell division.

Random copying errors are made during DNA replication.

DNA replication copies DNAnucleotides Every human cell

contains 3.4 billion nucleotide pairs.

DNA replication

Objectives of Forensic DNA Testing

To link an individual to a crime scene/criminal act.

To exonerate suspects. To identify victims of mass

disasters. To determine Paternity.

Specimen Samples used for Forensic

DNA Testing Blood (what components?) Semen Saliva Bones Skin scrapings Hair follicular tag

Main Procedures for DNA Fingerprinting:1. Isolation of the DNA to separate

the DNA from the cell.

2. Cutting w/ a restriction enzyme to make shorter base strands.

3. Sorting the segments by size, using electrophoresis

4. Analyzing the resulting print by identifying specific alleles/ genes.

DNA Fingerprinting Analysis:1. RFLP (Restriction Fragments

Lengths polymorphism)-use restriction enzymes to cut the DNA in fragments that are many diff lengths and shapes. The length vary from person to person. Restriction Enzyme-recognize a specific

sequence of bases & cut the DNA molecule at specific points.

Electrophoresis-a procedure that sorts/ separates DNA fragments by size.

Early 1980s: Restriction Fragment Length Polymorphism (RFLP)

Genetic variation in the distance between restriction enzyme sites

Template DNA digested by enzymes, electrophoresed, detected via Southern blotting

Power of discrimination in the range of 106-108 for a six probe analysis

Sir Alec Jeffreys

Mechanisms

for RFLPs

The Catch: RFLP testing requires a relatively

large amount of HMW DNA (~50ng = thousands of cells)

Not ideal for forensic evidence, in which small, degraded samples are common

2. PCR To The Rescue! Polymerase Chain Reaction =

molecular Xeroxing Three temperature phases, carried

out in a Thermal Cycler, replicate/ copy or “amplify” the desired DNA fragment(s)Dr. Kary Mullis

Eccentric Genius

PCR (cont’d) Works with lower quantity (1-2ng),

lower quality samples Power of discrimination goes from

102-106...not good enough for databasing

3. The Current Method of Choice: Autosomal Short Tandem Repeats

(STR) Non-coding, tetranucleotide

sequences which vary greatly from person to person in the number of repeating units

Requires <1ng of DNA to type 13-15 STR loci

Power of discrimination ranges from 1014-1023. World population is 109 so bring on the database!

Applied Biosystems 310 Genetic Analyzer

The Process In a Nutshell

Amplified DNA samples are injected into a capillary. Fluorescent tags on the DNA fragments are excited by a laser as they pass a window in the capillary, the fluorescence is recorded by a camera, and this signal is converted into a “peak” by the computer software.

X, Y,X Y

STR data

STR data (cont’d)

STR data (cont’d)

Item # AMEL D3S1358 vWA FGA D8S1179 D21S11 D18S51 D5S818 D13S317 D7S820 D16S539 THO1 TPOX

X, Y 17 8, 10

X, Y 17 8, 10

X 15, 17 11

X 15, 17 11

X 15, 17 11

X 15, 17 11

X, Y 17 8, 10

X, Y 17 8, 10

25(S)

25(E)

VICTIM

SUSPECT 9, 10 8, 9 9, 10

7, 8 11

15, 17 23, 26 14, 15 26 12, 15 10 9, 13

7, 8 11

16, 18 19, 26 1528, 32.2

8, 13 12 11, 12

14, 16 8, 13 12 11, 1216, 18 19, 26 1528, 32.2

14, 15 26 12, 15 10 9, 13 9, 10

00-00-00001V9780

8, 9 9, 10

STR TYPING SUMMARY SHEET

14, 16

15, 17 23, 26

Date: DNA Analyst / Serial #: DR #:

9/24/1999 MATTHIES

“The DNA profile obtained from Item 25(S) matches the DNA profile of the suspect. The combination of genetic marker types exhibited by Item 25(S) and the suspect occurs in approximately one in one hundred quadrillion (1017) individuals…”

How are these astronomical figures derived?

The product rule: combined probability of a series of independent events is determined by multiplying the probabilities of each event.

STR loci are inherited independently (unlinked)

Homozygous loci: p2 (same allele inherited from mother and father)

Heterozygous loci: 2pq (either allele could be inherited from either parent)

p(17)2 x 2p(15)q(17) x 2p(23)q(26)….

(.223)2 x 2(.083)(.25) x 2(.14)(.02) = .000013, which is equivalent to a probability of one in 76,000 using just 3 of the 13 loci!

STR Artifacts

-A (“minus A”): Incomplete addition of nucleotide ‘A’ by DNA polymerase; results in a peak that is one base pair smaller than allele peak.

STR Artifacts

Stutter: Slippage of DNA polymerase; results in a peak that is four base pairs (one repeat unit) smaller than allele peak.

STR Artifacts

Pull-up: Incomplete filtration of spectral overlap in fluorescent detection system.

Pull-up

DNA Mixtures

When more than one source of DNA is detected in a sample, assignment of genotypes becomes more difficult.

Degraded/Trace DNA Samples

Larger alleles “drop-out” when template DNA is low in quantity or quality, reducing certainty of genotypes.

The Combined DNA Index System (CoDIS)

A database of DNA profiles from violent felons and crime scene samples

Laws concerning who is eligible for the database vary from state to state

Database currently contains about 2,038,470 felons and 93,956 crime scene profiles (19,00 hits so far)

The Mystical Power of CoDIS

Extremely powerful investigative tool, linking crimes, and pulling suspects out of thin air!

Can prevent, as well as solve crimes!

The Dark Side of CoDIS(What the FBI doesn’t want you to know.)

DNA mixtures and degraded DNA profiles have lead to spurious matches

Stringent laws explicitly permit databasing innocent people

Adding arrestees to database violates presumption of innocence

However, the prosecution rate on case to offender matches is shockingly low! (~10%)

PCR

1. Isolate the DNA from crime scene evidence to get a pure sample and cut target strands.

2. Mix the DNA samples, primer, DNA polymerase enzyme & nucleotides in tube. Place in PCR chamber.

3. Heat the dsDNA to separate to 95°C.4. Cool to 50°C so that primers can attach to

ssDNA.5. Reheat to 72 °C so that DNA polymerase can

attach to end of primer.6. The DNA polymerase will now add

complementary nucleotides to the ssDNA. Two new strands are made.

7. Repeat cycle until desired # of DNA is copied.

Electrophoresis

1. Isolate pure sample & cut the DNA into fragments.

2. Make agarose gel.3. Add DNA with a buffer solution to wells in gel.4. Place the gel in the electrophoresis plate so

that a current passes through.5. The DNA will migrate from one end to the next

based on size.6. Stain the gel with Ethidium Bromide when the

electrophoresis is done.7. Place under UV light to observe band.8. Analyze/ compare bands for size comparison.

REview Take this scenario:

A woman gets out of bed, watches TV, talks on the phone, pets her cat, and then shoots her husband. She hides the gun and runs away. Which object would be most useful to the forensic serologist in finding out who the woman was?

A. the cat B. the phone C. the TV D. the TV remote E. none listed

What system used by the FBI compiles DNA of known violent offenders from all over the nation and can be used to match DNA with a sample found at a crime scene?

A. IAFIS B. IBIN C. SICAR D. CODIS E. DNAW

Review

Review: Who’s DNA is the unknown?

Suspect A Unknown Suspect B

Review

In this gel, the base-pair lengths are listed to the right. Which end of the DNA fingerprint was plugged to the NEGATIVE terminal during electrophoresis?

A. Top B. Bottom

C. Left D. Right

Review

In a talk-show scandal, it is revealed that one of three siblings has a different father.

GASP! Using the DNA fingerprint of just the mother, daughter and two sons, which sibling has a DIFFERENT father. A. Daughter B. Son 1 C. Son 2 D. all have the same

father—go figure!

Review

Which suspect's DNA matches the felon's DNA left at the crime scene?

A. suspect 1 B. suspect 2 C. suspect 3 D. none listed

Review

Whose the daddy? This mother is trying to decide between two men who desperately want to support her and her newborn baby. Both want to be a part of the baby's life, because they love the mother so much. Who gets the honor and privilege?

A. Dad 1 B. Dad 2 C. neither man

D. both

REview

Compare the sperm DNA to the two suspects and the boyfriend. Which male is most likely the assailant? A. suspect 1 B. suspect 2 C. boyfriend D. none listed

Review

In this DNA fingerprint, the blood sample taken from a crime scene is not the victim's and so is assumed to be the perpetrator. Which individual is the best suspect? A. Bob B. Sue C. John D. Lisa E. none

Review

There was a mix-up at the hospital. Which of these children belong to the parents?

A. All of the children

B. Children 2, 3 & 6 C. Children 1 & 5 D. Children 2 & 4