23 4. Agenda: 4/28 Objective: To determine how DNA is used in forensic cases Human Genome – how people differ DNA Uses and Sources DNA Fingerprinting

2 3 4

ACGCACTTCAGAACGCGTACTGACTGAA

TGCGTGAAGTCTTGCGCATGACTGACTT

Agenda: 4/28

Objective: To determine how DNA is used in forensic cases

Human Genome – how people differ

DNA Uses and Sources

DNA Fingerprinting – Steps needed

- Restriction enzymes

- Gel electrophoresis

- Polymerase Chain Reaction

Homework: Tuesday: Lab Notebook – with Serial Dilution & Spectrophotometer Wednesday: News article – specific and detailed for credit

Class notebookDate Assignment Pages

4/25 Processes represented in the Central Dogman

4/25 Proteins- function and structure

DNA Fingerprinting - Use of DNA in Forensic Cases

Restriction enzymes – background & lab preparation

DNA fingerprintingDNA forensics • Digesting a DNA sample

using restriction enzymes• Gel electrophoresis

– Process: running gels– Data analysis

• Polymerase Chain Reaction – Process – Interpretation

• Solving the Case • Paternity Case - Blacketts

What we need to know:

• RFLP – Restriction Fragment Length

Polymorphism

• Restriction enzymes• How/why does gel

electrophoresis works?• How/why does PCR work? • Use of informatics/statistics

The Human Genome

The sequence of bases make up our genes. The Human Genome Project determined the order of each of these

bases in all of our genes. Also found that most DNA is not coding for genes. There are many areas in which bases are repeated.

How many bases are there in the human genome?

a) 3,000b) 300,000c) 3 milliond) 3 billione) 3 trillion

Facts & Figures about DNA





a) 3,000b) 300,000c) 3 milliond) 3 billione) 3 trillion



3,000,000,000

We are not all exactly the same – What percent of your DNA is similar to any

other person in the world?




a) 99.9%b) 98%c) 90%d) 60%e) 10%





a) 99.9%b) 98%c) 90%d) 60%e) 10%




3 MILLION bases are different!

Forensic scientists focus on these variable regions to generate a “DNA fingerprint” for each individual


Summary – Nuclear DNA

DNA Use in Forensic Cases

• Most are rape cases (>2 out of 3)• Looking for match between evidence

and suspect• Must compare victim’s DNA profile

• Mixtures must be resolved

• DNA is often degraded

• Inhibitors to PCR are often present

Challenges

Human Identity Testing

• Forensic cases -- matching suspect with evidence

• Paternity testing -- identifying father

• Historical investigations• Missing persons investigations• Mass disasters -- putting pieces back together

• Military DNA “dog tag”• Convicted felon DNA databases

• YouTube – DNA forensics – 4 videos• https://www.youtube.com/watch?v=dXYztbkMXwU&list

=PLC0B027FC81C82602 – 2 minute overview

• Includes CODIS – Story• https://www.youtube.com/watch?v=VF5s1loHxx4&list=P

LC0B027FC81C82602• https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=P

LC0B027FC81C82602

• https://www.youtube.com/watch?v=yWKbQH2P6og&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=dXYztbkMXwU&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=dXYztbkMXwU&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=VF5s1loHxx4&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=VF5s1loHxx4&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=8w_VJ4G7qiw&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=yWKbQH2P6og&list=PLC0B027FC81C82602

https://www.youtube.com/watch?v=yWKbQH2P6og&list=PLC0B027FC81C82602

What are some sources of DNA?

Sources of Biological Evidence

• Blood• Semen• Saliva• Urine• Hair• Teeth• Bone• Tissue• Mucus• Ear Wax

DNA fingerprintingDNA forensics • Digesting a DNA sample

using restriction enzymes• Gel electrophoresis

– Process: running gels– Data analysis

• Polymerase Chain Reaction – Process – Interpretation

• Solving the Case • Paternity Case - Blacketts

What we need to know:

• RFLP – Restriction Fragment Length

Polymorphism

• Restriction enzymes• How/why does gel

electrophoresis works?• How/why does PCR work? • Use of informatics/statistics

Cutting the DNA with Restriction Enzymes

DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA.

Restriction Enzyme Digest

AGCTAGAATTCTTTACGCTCGGATGAATTCCACCTATCTCC

DNA can be cut into smaller pieces by restriction enzymes that recognize very specific sequences of DNA.

AGCTAG

AATTCTTTACGCTCGGATG AATTCCACCTATC

TCC

Restriction Enzyme Digest

AGCTAGAATTCTTTACGCTCGGATGAATTCCACCTATCTCC

Multiple Restriction Enzymes Exist for Cutting DNA

EcoRI GAATTC G AATTC

PstI CTGCAG CTGCA G

SmaI CCCGGG CCC GGG

HindIII AAGCTT A AGCTT

BamI GGATCC G GATCC

HaeIII GGCC GG CC

Separating the DNA fragments RFLP analysis

Visualizing the DNA Restriction Fragments 1 – Ladder to determine size (number of base pairs in each segment)

2-7 samples from suspects or victims

DNA Restriction Enzymes

• Evolved by bacteria to protect against viral DNA infection

• Endonucleases = cleave within DNA strands

• Over 3,000 known enzymes

Enzyme Site Recognition

• Each enzyme digests (cuts) DNA at a specific sequence = restriction site

• Enzymes recognize 4- or 6- base pair, palindromic sequences (eg GAATTC)

Palindrome

Restriction site

Fragment 1 Fragment 2

5 vs 3 Prime Overhang

• Generates 5 prime overhang

Enzyme cuts

Common Restriction Enzymes EcoRI

– Eschericha coli– 5 prime overhang

Pstl– Providencia stuartii– 3 prime overhang

The DNA DigestionReaction

Restriction Buffer provides optimal conditions

• NaCl provides the correct ionic strength

• Tris-HCI provides the proper pH

• Mg2+ is an enzyme co-factor

DNA Digestion

Temperature

Why incubate at 37°C?

• Body temperature is optimal for these and most other enzymes

What happens if the temperature is too hot or cool?

• Too hot = enzyme may be denatured (killed)

• Too cool = enzyme activity lowered, requiring

longer digestion time

Restriction Fragment Length PolymorphismRFLP

Allele 1

Allele 2

GAATTCGTTAAC

GAATTCGTTAAC

CTGCAGGAGCTC

CGGCAGGCGCTC

PstI EcoRI

1 2 3

3Fragment 1+2Different Base PairsNo restriction site

+

M A-1 A-2

Electrophoresis of restriction fragments

M: MarkerA-1: Allele 1 FragmentsA-2: Allele 2 Fragments

AgaroseElectrophoresis

Loading

• Electrical current carries negatively-charged DNA through gel towards positive (red) electrode

Power Supply

Buffer

Dyes

Agarose gel

AgaroseElectrophoresis

Running

• Agarose gel sieves DNA fragments according to size– Small fragments move farther than large fragments

Power Supply

Gel running

Analysis of Stained Gel

Determinerestriction

fragmentsizes

• Create standard curve using DNA marker

• Measure distance traveled by restriction fragments

• Determine size of DNA fragments

Identify the relatedsamples

Molecular Weight Determination

Size (bp) Distance (mm)

23,00011.0 9,400 13.0

6,500 15.0

4,400 18.0

2,300 23.0

2,000 24.0

100

1,000

10,000

100,000

0 5 10 15 20 25 30

Distance, mm

Siz

e, b

ase

pai

rsB

A

Fingerprinting Standard Curve: Semi-log

Polymerase Chain Reaction (PCR)

PCR can make many copies in a very short period of time

ACGCACTTCAGAACGCGTACTGACTGAATGCGTGAAGTCTTGCGCATGACTGACTT


Heat to 94°C: Denature Strands of DNA






Cool to 55°C: Allow primers to anneal

TGCGTGAA

TGACTGAA




Heat to 72°C: New DNA strand is synthesized




PCR can make many copies in a very short period of time














How do we generate a DNA fingerprint?

…After amplification of the variable regions through PCR

FBI’s CODIS DNA Database

Combined DNA Index System• Used for linking serial crimes and unsolved

cases with repeat offenders• Launched October 1998• Links all 50 states• Requires >4 RFLP markers

and/or 13 core STR markers• Current backlog of >600,000 samples

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

Overview

• Basic – DNA Fingerprinting – Overview: 6 min. Bozeman Science

Use of Short Tandem Repeats

• Non-coding sections (do not code from proteins)

• Inherited from parents – Individuals have 2

copies (alleles)

13 CODIS Core STR Loci with Chromosomal Positions

CSF1PO

D5S818

D21S11

TH01

TPOX

D13S317

D7S820

D16S539 D18S51

D8S1179

D3S1358

FGA

VWA

AMEL

AMEL

Short Tandem Repeats (STRs)

the repeat region is variable between samples while the flanking regions where PCR primers bind are constant

7 repeats

8 repeats

AATG

Homozygote = both alleles are the same length

Heterozygote = alleles differ and can be resolved from one another

Short Tandem Repeats

STRs are short sequences of DNA, normally of length 2-5 base pairs, that are repeated numerous times Example: the 16 bp sequence of "gatagatagatagata" would represent 4 copies of the tetramer "gata".

The polymorphisms (variations in DNA sequence between individuals) in STRs are due to the different number of copies of the repeat element that can occur in a population of individuals.

STR and probability

• Today's DNA profile :: DNA Learning Center

• http://www.dnalc.org/view/15983-Today-s-DNA-profile.html

http://www.dnalc.org/view/15983-Today-s-DNA-profile.html

http://www.dnalc.org/view/15983-Today-s-DNA-profile.html

STR Allele Frequencies

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)

Blacks (N=414)

Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

170 bp170 bp195 bp195 bp

Different primer sets produce different PCR product sizes for the same STR allele

TCAT repeat unitTCAT repeat unit

Multiplex PCR• Over 10 Markers Can Be

Copied at Once• Sensitivities to levels less than

1 ng of DNA• Ability to Handle Mixtures

and Degraded Samples• Different Fluorescent Dyes

Used to Distinguish STR Alleles with Overlapping Size Ranges

ABI Prism 310 Genetic Analyzer

capillary

Syringe with polymer solution

Autosampler tray

Outlet buffer

Injection electrode

Inlet buffer

Close-up of ABI Prism 310 Sample Loading Area

Autosampler Tray

Sample Vials

Electrode

Capillary

See Technology section for more information on CE

amelogenin

D19

D3

D8

TH01

VWA D21FGA

D16D18 D2

amelogeninD19

D3D8 TH01

VWA D21

FGA

D16D18 D2

Two

diff

eren

t ind

ivid

uals

DNA Size (base pairs)

Results obtained in less than 5 hours with a spot of blood the size of a pinhead

probability of a random match: ~1 in 3 trillion

Human Identity Testing with Multiplex STRs

Simultaneous Analysis of 10 STRs and Gender ID

AmpFlSTR® SGM Plus™ kit

STR genotyping is performed by comparison of sample data to allelic ladders

Microvariant allele

STR Allele Frequencies

0

5

10

15

20

25

30

35

40

45

6 7 8 9 9.3 10

Caucasians (N=427)

Blacks (N=414)

Hispanics (N=414)

TH01 Marker

*Proc. Int. Sym. Hum. ID (Promega) 1997, p. 34

Number of repeats

Fre

qu

ency

4 types of DNA

• http://learn.genetics.utah.edu/content/extras/molgen/index.html

http://learn.genetics.utah.edu/content/extras/molgen/index.html

http://learn.genetics.utah.edu/content/extras/molgen/index.html

When nuclear DNA is degraded:

Cases

• OJ Simpson• http://www.trutv.com/library/crime/criminal

_mind/forensics/serology/5.html• Australian• http://www.trutv.com/library/crime/

criminal_mind/forensics/serology/5.html

http://www.trutv.com/library/crime/criminal_mind/forensics/serology/5.html

http://www.trutv.com/library/crime/criminal_mind/forensics/serology/5.html

Blackett Family

• Paternity Case• http://www.biology.arizona.edu/

human_bio/activities/blackett2/overview.html

Documents

23 4. Agenda: 4/28 Objective: To determine how DNA is used in forensic cases Human Genome – how people differ DNA Uses and Sources DNA Fingerprinting