Unit 7DNA Forensics

Lesson 1 Introduction to Forensics

• Read the Powerpoint slides in Unit 1.• Respond to the questions at the end of the unit.• Class discussion of responses.

DNA Forensics • Every human carries a unique set of

genes.• The chemical structure of DNA is

always the same.• The order of the base pairs differs in

individuals.• Only 1% of DNA, about 3 million base

pairs, differs from person to person.• These variable regions can generate a

DNA profile unique to an individual.

DNA Forensics• Variable Regions of DNA• Protein coding sequences or genes produce a

protein product.• If there is a change in a DNA sequence, it may

change the sequence of amino acids and produce a mutant protein.• Non-protein coding sequences of DNA, some of

which have functions in the genome, do not produce protein.• Changes in these sequences are silent in terms of

traits.• However, there is a great deal of variability in

non-coding sequences, and DNA forensics primarily uses these non-coding sequences for DNA fingerprinting.

DNA Forensics

• Variable Regions• The locations of DNA sequences where

the genomes are different are called polymorphisms.• There are many polymorphisms in the

human genome. These have been identified and the positions of the polymorphisms on chromosomes have been determined.• The polymorphisms are referred to as

markers.• Markers can be a single base difference,

several base differences, or repeated sequences in the genome.

DNA Forensics

• The two types of markers frequently used in DNA fingerprinting are VNTRs (variable number tandem repeats or “vinters”) and STR (short tandem repeats).• Both markers are repeating base

sequences found in already determined positions in the human genome.

DNA Forensics• VNTRs• VNTRs are base sequences

approximately 10 -100 base pairs long that can repeat a few to many times.• VNTRs are different in each

individual and provide a scientific marker of identity.• There are about 29,000 VNTRs

scattered throughout the human genome. • http://vimeo.com/37851274

DNA Forensics• STRs• STRs are short base sequences , 2 – 9 nucleotides, that can repeat

approximately 5 -50 times.• STRs are dispersed throughout the genome.• However, the FBI uses 13 STR regions for analysis and comparison in its

library of DNA fingerprints. • This library is called the Combined DNA Index System (CODIS)

DNA Forensics

• FBI – STR loci used in testing.• http://vimeo.com/69239594

Lesson 1 What you need to know

• What makes DNA unique in each individual?• What sequences of DNA are of interest in DNA forensics.• Explain VNTRs.• Explain STRs.• Describe the relationship among the terms polymorphism, marker,

VNTR, and STR.

Lesson 2 RFLP

• Lecture: RFLP • Activity: - View video and respond to questions. - Complete RFLP simulation and explain the steps in the procedure. - Read handout and take notes for study purposes

DNA Forensics• Forensic Testing• There are two main types of forensic testing. RFLP (restriction

fragment length polymorphism) and PCR (polymerase chain reaction).• In RFLP testing, VNTRs and STRs can be tested. However, RFLP

testing requires large samples of intact DNA (which can be hard to find at some crime scenes).• PCR testing, on the other hand, is restricted to STR detection. PCR

requires little DNA and is still effective if the DNA is partially degraded. • http://www.youtube.com/watch?v=LLDa72sl8JA

DNA Forensics• RFLP• There are five basic steps to developing DNA profiles using VNTRs:• Extracting DNA.• Cutting DNA into fragments using restriction enzymes.• Separating the fragments based on size using gel electrophoresis.• Transferring the fragments to a nylon membrane (southern blotting), causing

immobilization.• Locating and identifying the fragments by applying a solution containing the probe of

interest, which then hybridizes to the immobilized DNA.• Probes will bind specifically to complementary VNTR fragments. Unbound and non-

specifically bound probe is removed using a washing process. The RFLP profile is then visualized by exposing the membrane to film or through the use of equipment, such as an imaging station.

DNA Forensics

DNA Forensics

• Once DNA is extracted, restriction enzymes are used to cut DNA into fragments.• The type of restriction

enzyme used can vary.• Restriction enzymes cut

the DNA in regions that flank the VNTR sequence.

DNA Forensics

• The number of repeats in the VNTR region varies in populations.• So fragments of various

sizes are created which are distinct in the individual being tested.• This is why it is called a

DNA fingerprint.

DNA Forensics• A gel electrophoresis is run.• At a VNTR loci, there are several alleles.• An individual will inherit some alleles

from their mother and some from their father.• These alleles have a different number

of tandem repeats.• When restriction enzymes cut the

flanking regions, the length of the repeat sequence determines the size of the DNA fragments.

DNA Forensics

• In addition to the VNTRs, the restriction enzymes will cut apart all of the sample DNA at the specific restriction site.• The gel electrophoresis will show

bands from all of the DNA.• A method called southern blotting

enables the scientist to select out the bands that represent the VNTR alleles.

DNA Forensics• DNA on the gel is transferred to a

membrane.• A radioactively labelled probe that is

complementary to the VNTR(s) of interest is applied.• The probe hybridizes (sticks) to the

matching VNTR sequences. • The membrane is placed on an X-ray

film and the scientists gets a picture of the radioactively labelled bands, each of which represented a different length of fragment

DNA Forensics

• A X ray film will show the different VNTR alleles as a banding pattern.• In the example on the right,

individual one has inherited allele B and C; one from each parent.• Individual 2 has inherited allele C

from both parents.

DNA Forensics – RFLP Activity

Watch the following video:• Forensic DNA Analysis

Respond to the following questions:• What does the DQA1 DNA test identify?• What are the advantages and disadvantages of this method?• Can the results of this test accurately pinpoint the source of the

blood? Why or why not?

DNA Forensics – RFLP Activity

Visit the following website:• Create a DNA Fingerprint

In your notebook, write down the name of each step in the procedure and provide an explanation of that step.

DNA Forensics – RFLP Activity Read the information in this handout:• How DNA Evidence Works

Outline the information in the handout for study purposes.

Lesson 2 What you need to know

• What are the two types of forensic testing and when are they used?• Explain the steps in the RFLP procedure?• Explain why there is variability in VNTR loci.• Describe southern blotting.• For what types of activities can RFLP be used?

Lesson 3 Crime Scene and Paternity Testing• Lecture: Crime Scene and Paternity Testing• Activity: DNA Fingerprinting

DNA Forensics

• Crime Scene Forensics• Any time two people make physical contact, some biological material

is transferred.• At a crime scene, forensic investigators will collect this evidence (ex.

hair, skin, blood, semen).• The evidence undergoes DNA testing to establish a DNA profiles of

the suspect.• If the suspect is unknown, law enforcement can search the FBI

database CODIS which stores DNA profiles of previously convicted offenders.

DNA Forensics

• Once a suspect is located, a cheek swab is taken and the DNA is extracted for forensic testing.• A match occurs if the suspect’s

DNA profile matches the evidence DNA profile.• A match, however, does not

prove the suspect is guilty.

DNA Forensics

• Sometimes a DNA match is coincidental because a close relative may have committed the crime and has the same VNTR alleles as the suspect.• The criminal could have been an identical twin.• There may have been errors in testing or evidence could have been

compromised.• Law enforcement needs to rely on witness testimony and

circumstantial evidence as well as the DNA evidence.

DNA Forensics

• Paternity Testing• Individuals inherit one chromosome from their

mother and one from their father for each of the 26 chromosomes.• When reading the banding pattern, one band

from the child MUST match a band from the father or the mother. If the child’s band does not match one of the adults, then they are not the parent. • http://

www.sumanasinc.com/webcontent/animations/content/paternitytesting.html

DNA Forensics

• Refer to your handout for directions.• Solve the paternity and crime scene problems.

Lesson 3 What you need to know

• Describe how to read a gel banding pattern for crime scene investigations.• Why does a DNA match not necessarily mean a suspect is guilty?• Describe how to read a gel banding pattern for paternity testing

Lesson 4 Laboratory Southern Blot Testing• Refer to your lab handout for laboratory procedures and questions.

Lesson 5 PCR Forensic Testing

• Lecture: PCR and STR Testing• Activity: Tutorial STR DNA Profile Analysis Blackett Family Pedigree

PCR Forensic Testing• Kary Mullis’ invention, in 1983, of the polymerase

chain reaction (PCR) won him the Nobel Prize in Chemistry. • This invention, together with the discovery in the

late 1980s of short tandem repeats (STRs) – 2-9 bp repeated sequences, also called microsatellites – paved the way for the high-speed genetic fingerprinting technique that forensic scientists use today.• PCR enables a DNA locus of interest to be amplified

exponentially, generating a billion copies of a single DNA molecule in a few hours.

PCR Forensic Testing

• For PCR analysis, STRs flanked by sequences that are identical in all human beings are needed (sequences are conserved).• Then use fluorescent primers – short DNA molecules that are

complementary to the conserved flanking sequences (genes 1134 and 1135 in the figure above) – to initiate the PCR.

PCR Forensic Testing

• Once the DNA has been amplified, it can be separated either by gel electrophoresis or, in modern forensic science, by electrophoretic automated sequencing, and can be visualized as a genetic fingerprint.

PCR Forensic Testing – Gel Electrophoresis

• There are two copies of each chromosome, so there are also have two copies of each STR.• If, for each copy of the STR, there the same number of repetitions (i.e. the same

allele), the PCR analysis reveals only one size of DNA fragment: the person is homozygous for that STR allele (green arrow in the above picture ). • If the two chromosomes carry non-identical alleles for that STR, there are two

sizes of fragment: the person is heterozygous (red arrow in picture).

PCR Forensic Testing

• The blue arrows indicate two people who are heterozygous and have the same number of repeats for each allele at the STR locus; this means that they cannot be distinguished by the fingerprint. • They may be twins, but it is also likely that two unrelated persons will have the

same number of repeats if only one STR is analyzed .

PCR Forensic Testing• If only one STR is analyzed, the chance of two

unrelated people having the same PCR-based genetic fingerprint is high – between 1:2 and 1:100 • This is because STRs have fewer alleles and

lower heterozygosity than the VNTRs used in RFLP-based genetic fingerprints.• To overcome this disadvantage, multiple STRs

are analyzed simultaneously; with 13 STRs used in forensic casework in the USA, a power of discrimination of 1 in hundreds of trillions can be achieved .

PCR Forensic Testing – Electrophoretic Automated Sequencing• Amplified PCR products undergo

automated testing with capillary gel electrophoresis.• During PCR, primers that are fluorescently

labeled are used during the amplification.• Enzymes then separate the DNA strands

which are then subjected to gel electrophoresis procedure using thin capillary tubes instead of agarose gels.

PCR Forensic Testing

• As the DNA migrates through the capillary tube, a laser reads the wavelength of light for the period of time it takes for the primers to migrate past the laser.• The migration rate correlates to the molecular weight of the

molecule. Larger molecules(those with more repeats) take longer to migrate past the laser.• A computer then prints out the molecular weights of the STR alleles

in a format called an electopherogram.

PCR Forensic Testing

• Electropherogram of a woman, generated by multiplex PCR and subsequent electrophoretic automated sequencing.• Eight STRs (D3, TH01, D21, D18, SE33, vWA, D8 and FGA) and amelogenin

(which indicates the sex) were analyzed. • The blue, green and black curves represent amplified STRs (with repeat

numbers below the peaks). The red curve is the marker (DNA fragment size labelled in bp)

PCR Forensic Testing• http://www.youtube.com/watch?v=x6U7JKpG2Gw#aid=P-5YIollEYoShort Tandem Repeats

Lesson 5 STR Activity

• Visit the following website• http://

www.biology.arizona.edu/human_bio/activities/blackett2/overview.html• Read: The science of STR DNA Profile Analysis• Complete the student activity

Lesson 5 What you need to know

• What is a microsatellite?• What is needed to begin the PCR process in order to identify the

STR?• Describe how to read STRs using gel electrophoresis.• Why are multiple STRs analyzed at the same time?• Explain how electrophoretic automated sequencing works.• How do you read an electropherogram?

Lesson 6 Barcoding

• Activity: DNA Surveillance Unit: Is That An Endangered Whale You Are Eating?

Barcoding• Visit the following website:• http://

teachingbioinformatics.fandm.edu/activities/dna-surveillance-unit-endangered-whale-you%E2%80%99re-eating

1. Read: What is DNA Barcoding.2. Read: Fish Tale – has a DNA Hook3. Complete Lab #1 and Lab # 2Corrections for Lab #1 and Lab#2Main site for labshttp://dna-surveillance.fos.auckland.ac.nz:23060/Food Inspector- Lab #2 http://dna-surveillance.fos.auckland.ac.nz:23060/page/food/title

Lesson 6 What you need to know

• What is barcoding?• What is the gene region used in barcoding and why is it useful?• What are 2 applications of barcoding?

Lesson 7 Human Migration

• Lecture: Mitochondrial DNA and Y chromosome – Human Migration• Movie: The Journey of Man

Human Migration

• Molecular Clocks• Human migration can be tracked by tracking DNA

sequence mutations.• Any DNA sequence with a known mutation rate can

serve as a molecular clock to determine how far back in time an investigator must go to find a common ancestor in a given geography.• Scientists can determine the number of differences in

the DNA sequence to calculate the most recent common ancestor (TMRCA) for two populations.

Human Migration

• To calculate TMRCA, scientists look at DNA changes in populations over time.

• Populations A and D represent different groups of people living in different geographic areas.

• The people that A and D descended from are the same population.

• Populations B and C split off from A and migrated to two different areas.

Human Migration• When A and D first settled, they

had similar DNA sequences.• Over time, mutations occurred and

the DNA sequences became less similar.• The later generations of A migrated

to form populations B and C.• Over time, both B and C

accumulated genetic changes that were different from each other.

Human Migration• If scientists have a known genetic

marker with a known rate of mutation, they would conclude that populations A and D have much DNA similarity with few differences.• Populations B and C, although

different, would have much in common with population A.• Thus, they could trace the most

recent common ancestors.

Human Migration• Genetic Markers• The two genetic markers most frequently used in human migration

studies are mitochondrial DNA (mtDNA) and the Y chromosome.

Human Migration

• Mitochondrial DNA• mtDNA contains 13 protein encoding genes, 22 tRNAs, and 2 rRNAs.• Mitochondria are present in large numbers in the cell so much DNA can

be isolated.• mtDNA mutates at a higher rate than nuclear DNA. Differences

between closely related individuals can be resolved.• Mitochondria are inherited only from the maternal line. A direct

genetic line can be traced.• mtDNA does not recombine like nuclear DNA during meiosis thus

creating a clearer genetic history.

Human Migration• Study of mitochondrial DNA sequences

indicate that modern humans arose from a few females about 171,500 years ago.• At this time, mitochondrial sequences

coalesced into one.• The composite female is referred to as

mitochondrial Eve.

Human Migration

• Y Chromosome• The Y chromosome is passed from father to son and enables a clear

genetic history to be established.• Recombination can occur during meiosis in males between the X and

Y chromosome but only at the ends of the Y chromosome because X and Y are unmatched. Thus a large part of the Y chromosome is conserved and a direct genetic line can be traced.• There are very few genetic changes on the Y chromosome but small

polymorphisms do occur.• There are SNPs and STRs on the Y chromosome.

Human Migration

• Based on DNA analysis of the Y chromosome, all males are descended from a single male who live 35,000 – 90,000 years ago.• This individual is referred to as Y

chromosomal Adam.

Human Migration

• The origin of modern humans is a subject of significant controversy.• There are, however, several points of agreement.• Homo sapiens evolved from Homo erectus, a human like

primate who walked upright.• H. erectus migrated out of Africa 2 million – 1 million years

ago into Europe and Asia.• Another wave of migration took place 100,000 – 200,000

years ago.• The more recent population encountered H. erectus as they

migrated into Europe and Asia, and they coexisted.

Human Migration

• The debate about the evolution of H. erectus into H. sapiens involves three competing theories.

1. Uniregional model (African replacement)2. Multiregional model3. Assimilation model

Human Migration• Uniregional (African replacement)• The more recent African migrants did

not interbreed with other hominids in Europe or Asia.• Instead the other hominids became

extinct and the more recent migrants replaced them because they were better able to survive.• These migrants were the sole

forerunners of modern humans.

Human Migration• Multiregional• Proposes the original H. erectus

migrated into Europe and Asia and then evolved into H. sapiens.• Multiregional evolution involved

significant interbreeding among the more recent migrants and other hominid groups.• In each region, the characteristics

of modern humans emerged.

Human Migration

• Assimilation • Recent migrants did

interbreed with some other hominid groups but the degree of interbreeding varied in different geographic regions and from one time period to another.

Human Migration

• mtDNA and Y chromosome studies support the Uniregional model.• However, critics argue that

the picture is incomplete and autosomal DNA needs to be studied.

Human Migration

• Movie : The Journey of Man (150 min.)• http://www.youtube.com/watch?v=Cf7EcSkYivQ

Lesson 7 What you need to know

• Describe how a molecular clock can show the most recent common ancestor.• Why is mtDNA a good genetic marker for human migration?• Why is the Y chromosome a good genetic marker for human

migration?• Explain the uniregional, multiregional, and assimilation models for

the evolution of modern humans.