1

• Lecture 2: DNA analysis

• Lecture 3: Bloodstain Pattern Analysis

• Lecture 4: Fingerprints

Forensic Biology

Dr. B. L. Lim

Associate Professor

School of Biological Sciences

bllim@hku.hk

Tel: 22990826

Announcements

1. Collect hair/fiber for the lab in advance!

2. Carry hair/fiber to the lab in a plastic bag (Sept. 27 or Oct. 4,

9:30 - 12:30 pm)

1. Venue: Lab C/F/G/H, 1st Floor, KBSB building

2. Draw the patterns on the lab report 1 (Part I)

3. Tape the hair/fiber on report 1 (Part I)

4. Submit report 1 (Part I) immediately after the lab

5. Submit report 1 (Part II) after the lecture on 9 Oct.

II. Quiz 1

1. Date: During the lecture on 2 Oct (Wed)

2. Time: 15 mins

3. Format: 10 MCQ

4. Covering topics in Forensic Biology (lectures 2 - 4)

5. 7.5% of final grade

6. Bring calculator with Trigonometric function (No mobile phone please!!)

I. Practical 1

Outline

• 1. Introduction to Forensic DNA analysis

• 2. The development of forensic DNA testing

methods

• 3. Searching for biological evidence at scene

• 4. Forensic genetics and method

• 5. Result interpretation and evaluation of DNA

evidence

• 6. Case sharing and discussion

Recommended book

Fundamentals of Forensic DNA Typing, John M. Butler,

Elsevier Academic Press.

First use of forensic DNA testing to

convict a murder

• Two young school girls were raped and murdered

in a small town in UK (1983, 1986)

• Semen was identified to be type A of ABO blood

types and PGM 1+ (10% local population were of

these types). Both were from the same man.

• Blood typing results cannot be used to identify a

suspect. It can only be used to “exclude” a suspect

• Suspect Richard Buckland admitted to be the

murderer of one of the girls.

First use of forensic DNA testing to

convict a murder • Prof. Alec Jeffreys was invited to perform DNA analysis

(RFLP multi-locus probes)

• Richard Buckland, a 17-year-old with learning disabilities, was the first person in the history proved to be innocent by DNA testing

• Over 4000 blood samples of male adults, aged 17-34, from three local villages were collected for DNA analysis.

• In a pub, the owner heard from a conversation that a baker gave his blood to police for examination, but under the name of his colleague, Colin Pitchfork, for £200.

• The murderer Colin Pitchfork was the 4583th blood donor in 1987. He

2

Conventional blood grouping

• Based on antigens on RBCs (i.e.. ABO blood groups) and electrophoretic polymorphisms of plasma proteins (e.g. Gc grouping) and red cell enzymes (e.g. PGM grouping).

• Can only be used to EXCLUDE a suspect

• Low power of discrimination

• almost 40% of local Chinese population are of type O of ABO blood types; the rarest type is type AB (6%)

• Unable to detect degraded samples

• Low sensitivity. e.g. unable to detect limited amount of samples such as epithelial cell debris from hands or even tiny bloodstains.

Forensic DNA analysis

1. High power of discrimination

-identify/differentiate an individual

2. Able to analyze degraded samples

3. Highly sensitive

- require very limited amount of samples

4. Possibility for automation

- avoid human error *

(Murphy’s Law: everything that can go wrong will go wrong)

- possible for high throughput analysis (i.e. many samples can be analyzed at the same time)

- save human resource

Forensic Science

•Identify the Victim/Remains

•Identify the Criminal (Murderer/Rapist…)

Evidences for Courts

•Who is the victim?

•What injuries are present?

•When did the injuries occur?

•Why and how were the injuries produced?

Forensic Pathology

Forensic Odontology

-Teeth examination

-Facial reconstruction

Source of photo: southwalespolicemuseum.org.uk

Dr. Edmund Locard (1877-1966)

The Sherlock Holmes of France

Locard Exchange Principle

With contact between two

items, there will be an

exchange

Searching for Evidence

• Every contact leaves a trace (Edmund Locard)

• Transfer of evidence

person to person

person to object

object to object

3

Evidence

Physical

identification

Chemical

identification

Biological

identification

Searching for Evidence

• Physical evidence

Shoe print, glass fragments, fiber and etc

• Biological evidence

Bloodstains, semen stains, saliva, hair and etc

Precaution of Exhibits Collection

• Avoid contamination

• Wear clean latex gloves for collecting evidence items/exhibits

• Exhibits must be packaged separately into paper bags, and properly marked with case number, item number, collection date, and initialed across the package seal in order to maintain chain of custody which requires proper documentation to prove the chain of possession from the time of collection until entered in evidence in court

Collection of Exhibits

• Small exhibits

Cigarette end

• Special exhibit

A bottle of drinking water without the cap

Evidence

Physical

identification

Chemical

identification

Biological

identification

Forensic Biology

What are useful biological

materials?

4

Forensic Serology

A Bloody Mess

Find a Stain at a Crime Scene

•Is it Blood?

•Is it from human or not?

•Blood Typing

- A, B, AB, O

Is it blood?

• Presumptive test

- very sensitive

- can have false positive

• Confirmatory test

- not as sensitive

- specific to hemoglobin

1. Phenolphthalein color test

- based on oxidizing activity of hemoglobin

H2O2 H2 + [O] color

- turns pink if it is blood, remain colorless

if blood is not present

Fe3+[heme]+

Non-Human Blood?

Presumptive test

5

2. Hemastix®

strips

Non-Human Blood?

Blood?

Detection of

heme of

hemoglobin

3. Luminol – Fluorescent blood detector

How does Luminol detect blood?

Reacts with heavy metals

to glow in the dark. e.g.

iron, copper, chlorine

H2O2 + AH2 2H2O + A

Fe in Heme

Even at areas cleaned after a murder! Confirmatory Tests

Takayama and Teichmann tests

Chemicals that form crystals

with hemoglobin

Highly specific

Examine under

microscope

6

If it is blood, is it

human blood?

Any test specific to human sample?

Test sample

Rabbit serum that has

been sensitized to human

blood.

• Precipitin test

antigen + antibody

= precipitate

•Species Identification tests (Ouchterlony Gel)

Anti- Anti-

OK! So it is human blood!

But, so ?

Blood groups?

7

The Nature of Blood

1. Plasma (55%)

2. Blood solids

Red blood cells White blood cells Platelets

• In 1901 Dr. Karl Landsteiner

A - A antigen only

40-42% population

B - B antigen only

10-12% population

AB - Both A and B antigens

3-5% population

O - Neither antigen

43-45% population

Antibody and Agglutination

- Human Blood group determination

8

Microscopic view

of normal red blood

cells

Microscopic view of

agglutinated red

blood cells

Other proteins in Blood:

- Rh factor - 85% population

- Polymorphic enzymes

Phosphoglucomutase

PGM1 – 58%

PGM2 – 6%

PGM2-1 – 36%

Adenosine deaminase

Haptoglobin

Transferrin

…………….

Matching of Blood Type and Isozymes

Question: What is the possibility of a suspect’s blood sample sharing the

following characteristics with the blood sample collected at the crime scene ?

• Blood Group A

• Basic subtype A2

• Protein AK

• Enzyme PGM 2

42%

25%

15%

6%

Answer: 0.00094 (0.42 X 0.25 X 0.15 X 0.06) or

1 in 1063

DNA. What it is …… Only 4 base type in DNA, but its sequence does vary

9

Look at the neighbor to your left and

to your right. You're 99.9% identical.

Why DNA?

- DNA is the building block detailing

hereditary information

- individually unique (except monozygotic

twins)

- present in virtually all body tissues

Blood

Teeth

Saliva

Tissue

Bone

Hair

Semen

Sources of DNA

How much DNA is needed for forensic analysis?

1 nanogram (ng)

1 ng = 10-3ug = 10-6 mg = 10-9 g

10

What should we compare?

DNA Polymorphism (“many forms”)

-Regions of DNA which differ from person

to person

- 99.9% of DNA is the same in all

humans.

- Only 0.1 % of DNA (3 million

bases) differs from one person to

another.

DNA Technologies used in Forensic Science

1. Restriction fragment length polymorphism (RFLP)

2. Polymerase Chain Reaction (PCR)

3. Short Tandam Repeats (STR)

4. PCR-STR

Basic forensic genetics

• Human body is comprised of many cells.

• Each cell contains DNA (except red blood cells).

• The chromosomes in each cell are identical.

• Chromosomes are comprised of many genes (or

alleles).

• Genes are in pair; one comes from our mother and

another from our father.

• Forensic DNA analysis is to measure the length of

these pair of alleles.

Child

Father Mother

11

Restriction fragment length polymorphism (RFLP)

Rules of Inheritance

The DNA Fingerprinting Process

1. DNA extraction

2. Restriction enzyme

digestion (To cut)

3. Gel electrophoresis

(To separate)

4. Transfer of DNA to a

filter

5. Hybridization with a

specific DNA probe

(To bind)

6. Autoradiography (X-

Ray film, detect)

RFLP

Kary B. Mullis

Polymerase Chain Reaction (PCR)

1993: Nobel Prize for Chemistry

PCR Amplification

Patent sold for US$300M in 1991

引物

DNA polymerase

dATP, dTTP, dGTP, dCTP 10 - 20 ul

樣品 DNA/模版 DNA

Buffer 緩衝劑

Reagents required for PCR

Primer

Sample DNA/template DNA

12

PCR • Denaturation 95℃

• Annealing 60℃

• Extension 72℃

dsDN

A

ssDNA

dsDNA

新鏈 Taq DNA polymerase

dATP, dTTP, dGTP, dCTP

Primer

引物

引物 dNTP

New strand

Polymerase Chain Reaction

36 cycles theoretically will produce = 236 = 60 billions copies

– Amplify one molecule of DNA into billions

of copies in a few hours

– Identifies an individual based on

microscopic amount (1 ng) of evidence

Much more sensitive than RFLP!

HK Government Lab Short tandem repeats (STR) or

Variable number tandem repeats (VNTR)

1985 – Alec Jeffreys discovers multilocus VNTR probes

Repeats of 4-6 nucleotides

13

15 CODIS Core STR Loci with

Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

PCR-STR typing analysis

• STR stands for short tandem repeat.

AAGTAAGTAAGTC AAGT one repeat unit

TTCATTCATTCAG TTCA

• PCR-STR typing is to measure the length of this pair of repeat sequence at a particular locus.

• This pair of repeat sequences is the alleles. They are usually of different length.

1. DNA extraction

2. PCR

- Specific primer pair for each loci

- Each primer pair linked with a specific fluorescence probe

- Each primer pair produces PCR products within specific size

range

3. Separation and Detection of the PCR products by capillary

electrophoresis and laser detection, respectively.

Three steps of PCR-STR Instrument for STR Analysis

• Amplified DNA

loaded into

autosampler

• DNA migrates

through capillary

• DNA size analyzed

by computer

Capillary Electrophoresis + Laser Detection

Example: All possible alleles in 3 STR loci

Markers

Crime Scene

Suspect 1

Suspect 2

Suspect 3

Suspect 4

14

If all 15 core loci are

genotyped, probabilities of 1: X

quadrillions (1015

) can be found.

How did the forensic scientist figure out the blood stain on the

van were from two individuals?

Criminal + Victim

For an individual, no more than 2 alleles should be found at each locus.

One from mother, one from father!

1. Set up in 1998 by FBI

2. Links all 50 states

3. Two PCR with 15 STRs

4. 2 DNA databases

- Convicted Offenders Index

- Forensic Index (Crime scene)

Effective – Repeat Offenders

5,000,000 profiles

188,000 profiles

As of Aug, 2007

“The Criminalistics and Quality Management Group

carries out various types of laboratory analysis and

conducts crime scene examinations. A total of 1871

cases were attended in 2011(628 in 2004). The number

of exhibit items submitted for forensic DNA analysis

increased sharply from 4846 in 2004 to 9059 exhibit

items. At the end of 2011,the number of DNA data

stored within the database had increased to 39,949.

The DNA Database Section regularly compares new DNA

data with those in the DNA database at two levels:

(a) convicted offenders' DNA profiles (b) between

outstanding profiles from unsolved cases. In 2011

there were respectively 150 and 17 pairs of matches,

providing important investigative leads to the

Police Force.”

(HKSAR 2011 Yearbook)

HKSAR Government Lab

DNA database: since Jan 2001

Exonerate innocent persons wrongly accused of crimes

15

Mix-up man unlikely to trace biological parents (STANDARD) 02月 25日 (2008)

「一日阿媽，終生阿媽」 32年前調錯嬰 尋親只為求真相 (明報) 02月 24日

A 31-year-old man at the center of Hong Kong's first baby mix-up case in local

public hospitals may not be able to find his biological parents, a Hospital

Authority spokesman said yesterday.

Establish paternity and other family relationships

11 Sept., 2001 26 Dec., 2004

Identify crime and catastrophe victims

The number and identity of the victims were unknown.

20,000 pieces of human remains from the World Trade Center

site.

Remains of only 1585 of the 2792 known victims were identified.

Died in prison and Rumor of Escape of King Louis XVII

Louis-Charles Louis XVI Marie Antoinette

1789 French Revolution

1795 Louis Charles died in prison

Heart stolen by a doc during autopsy

2000 DNA test of the heart

2004 Buried the heart in the royal cemetery with

his parents after 209 years

End of Plantagenet Dynasty (1154-1485)

Beginning of Tudor Dynasty (1485-1603)

Battle of Bosworth Field (22 Aug 1485)

Henry Tudor, Earl of Richmond, the first English monarch of

the Tudor dynasty, defeated Richard III, the last king of the

House of York.

In 2012, an archaeological excavation was conducted on a city council car park using

ground-penetrating radar on the site once occupied by Greyfriars, Leicester. The

University of Leicester confirmed on 4 February 2013 that a skeleton found in the

excavation was, beyond reasonable doubt, that of Richard III.

Please bring along with you an electronic calculator with Trigonometric

function and a ruler with scale in mm.

Next lecture and quiz

Dr. Henry Cheung, former Government Lab Scientific Officer.

Acknowledgement