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

• 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 nonspecifically 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
• 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/anim ations/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

• 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.

• 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 .

• 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 .

• 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.

• 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-5YIollEYo
Short Tandem Repeats

Lesson 5 STR Activity
• Visit the following website
• http://www.biology.arizona.edu/human_bio/activities/blackett2/over view.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-unitendangered-whale-you%E2%80%99re-eating
1. Read: What is DNA Barcoding.
2. Read: Fish Tale – has a DNA Hook
3. Complete Lab #1 and Lab # 2
Corrections for Lab #1 and Lab#2
Main site for labs http://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 model
3. 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.