FINAL DNA FORENSIC EXAM (DNA FORENSIC EXAM 1, DNA FORENSICS EXAM 2, DNA Forensics Exam 3)
Explain the process of PCR, with specific reference to: the requirement for primers, DNA polymerization, DNA polymerase enzyme and temperature cycling profiles
)PCR-makes a lot copies of particular fragment of DNA •main activity is polymerization of nucleotide bases to form a new strand of DNA energy for DNA polymerization come from hydrolysis diphosphate bond(dNTP) Denature(separates 2 strands) 96C | | Annealing (add primers) 55C | | Extension(elongates primer polymerase taq) 72C
Describe how to move an STR locus by changing the fluorescent dye
-Allow automated detection -Come in different colors, allows you to detect different PCR products in the same lane, even if they are the same size
Explain the actual purpose for which a manufacturer provides an allelic ladder
.Provide a standard that practicing laboratories can use for validation studies
List and explain the limitations of RFLP markers for use in forensics
1. Subjective interpretation 2. A lot of DNA is required
Explain the advantages of STR loci, compared to RFLP loci, for forensic analysis
1.) •Multiple alleles, each at about the same frequency in the population 2.)•Inherited as simple Mendelian alleles 3.)Easy to visualize 4.) Scorable without ambiguity RFLP loci can only do 1 and 2(+/-)
A mother's genotype at CSF1PO is 9/12. The father's genotype is 9/10. Which of the following is a possible genotype for anyone of their children? 1.9/9 2.9/10 3.9/12 10/10
1.9/9 2.9/10 3.9/12
Describe the searches that are performed within the CODIS system and explain the kind of information that can be gained from a match in each type of search
1.The Forensic Index is compared to the Offender Index -A hit provides an investigative lead to a known-identity suspect 2.The Forensic Index is compared to the Missing Persons Index -We will discuss this when we cover missing-persons investigations 3.The Forensic Index is compared to itself -Connect previously unconnected crimes
Describe the basic principles behind visualization of DNA fragments by Southern blot hybridization and autoradiography
1.Transfer the DNA from the gel to a solid surface ("Southern blot"), so the DNA cannot move around 2. Break the hydrogen bonds to make the immobilized DNA single-stranded (NaOH) 3.Label a 'probe' with radioactivity and hybridize to the DNA on the membrane 4.Add a solution containing radioactive labeled probe(also single-stranded) 5.Expose to X-ray film
DNA polymerase enzyme requires ___________ in order to initiate elongation of a DNA molecule. 1.a single-strand - double-strand boundary 2.a brief time at 94° 3.an RNA primer 4.ATP
1.a single-strand - double-strand boundary
A single-locus RFLP probe can be made by 1.designing an oligonucleotide probe that is complementary to the flanking sequence. 2.designing an oligonucleotide probe that is complementary to the repeat region. 3.designing an oligonucleotide probe that is complementary to a protein-coding gene. 4.finding a minisatellite sequence that only occurs once in the genome. 5.placing a radioactive label directly on the genomic DNA.
1.designing an oligonucleotide probe that is complementary to the flanking sequence.
The ultimate goal of DNA forensic analysis in a criminal case is to provide evidence that will 1.support determination of the guilt or innocence of the defendant. 2.determine the identity of the source of a biological specimen. 3.determine the probability that a person chosen at random would have the same genotype as that of a biological specimen. 4.determine the DNA sequence of a biological specimen.
1.support determination of the guilt or innocence of the defendant.
If a minisatellite repeat unit exists in many places in the genome, a probe that will show allofthose locations should be complementary to 1.the repeat unit. 2.the flanking region. 3.the restriction enzyme site. 4.a random sequence, as long asit is unrelated to the repeat unit. Can't be done, so "none of the above
1.the repeat unit.
Describe the basic principles behind separation of DNA fragments using electrophoresis
1: Cut the genomic DNA with one or more restriction enzymes 2: Sort the digested genomic DNA by size, using electrophoresis
AA Aa aa 0.09 0.62 0.29 What is the allele frequency of A? 1.0.04 2.0.4 3.0.03 4.0.3 5.Not enough information is provided to determine the allele frequencies
2.0.4
A = 0.7 a = 0.3 The population is at H-W equilibrium.What is the genotype frequency of AA? 1.0.14 2.0.49 3.0.049 4.0.42 5.Not enough information is provided
2.0.49
What is the main source of variation between alleles at an RFLP locus? 1.Single-nucleotide mutations in the repeating unit. 2.Single-nucleotide mutations in the restriction enzyme recognition site 3.Differences in the number of copies of the repeating unit. 4.Single-nucleotide mutations in the flanking region. 5. Differences in the number of copies of the flanking region
2.Single-nucleotide mutations in the restriction enzyme recognition site 3.Differences in the number of copies of the repeating unit.
Which of the following is NOT an error were made in the prosecution of Sally Clark for the murder of her two sons? 1.The court allowed a pediatrician to testify as an expert in statistics. 2.The court allowed the prosecution to add the probabilities together, when they should have been multiplied . 3.The event that a second child in the same family would die suddenly of natural causes was assumed to be independent of the event that the first child had died suddenly of natural causes. 4.The prosecution was allowed to argue that the chance of two sudden deaths in the same family was so low that the deaths must have been an act of murder.
2.The court allowed the prosecution to add the probabilities together, when they should have been multiplied
The "Prosecutor's Fallacy" is when 1.the prosecutor relies on eyewitness testimony. 2.The prosecutor argues that the 1-in-a-million chance of a random match means that there is a 99.9999% chance the defendant is guilty. 3.the prosecutor tells the jury that the defense lawyer was previously convicted of a crime, so the defense is not credible. 4.the prosecutor tells the jury to ignore the defense argument that there is expected to be 100 other people in the city that also match the description. the prosecutor claims that the defendant's alibi is a fantasy.
2.The prosecutor argues that the 1-in-a-million chance of a random match means that there is a 99.9999% chance the defendant is guilty.
The important difference between alleles at a VNTR locus is 1.a point mutation in the DNA sequence. 2.a difference in the number of copies of the repeat unit. 3.an inversion of the chromosomal DNA. 4.a difference in the temperature used for hybridization.
2.a difference in the number of copies of the repeat unit.
At 60°C 1.primers will anneal. 2.some primers will anneal, others won't 3.Taqpolymerase has no activity 4.Taqpolymerase has its maximum activity
2.some primers will anneal, others won't
At 72°C 1.primers will anneal. 2.some primers will anneal, others won't 3.Taq polymerase has no activity 4.Taq polymerase has its maximum activity
2.some primers will anneal, others won't 4.Taq polymerase has its maximum activity
For a marker with 4 alleles: A1(p), A2(q), A3(r), A4(s)IF: p=0.2, q=0.4, r=0.1What is s? 1.0.5 2.0.4 3.0.3 4.0.2 5.We need more information to answer that
3.0.3
A1A1 A1A2 A1A3 A2A2 A2A3 A3A3 0.01 0.02 0.32 0.30 0.10 0.25What is the allele frequency of A2? 1.0.30 2.0.40 3.0.36 4.0.5 5.Not enough information is provided to determine the allele frequencies
3.0.36
A = 0.7 a = 0.3 The population is at H-W equilibrium.What is the genotype frequency of Aa? 1.0.21 2.0.49 3.0.42 4.0.09 5.Not enough information is provided
3.0.42
Why can DNA grow only from 5' 3'? 1.Because the free nucleotides are all in the 5' 3' orientation. 2.Because DNA does not have the 2'-OH that RNA has. 3.Because of where the energy for polymerization comes from. 4.Because 3' 5' DNA would have the nucleotides on the outside and the phosphates on the inside. Because 3' 5' DNA cannot be translated correctly
3.Because of where the energy for polymerization comes from.
Why do PCR reactions require primers? 1.So the polymerase knows where to start 2.So the polymerase knows which direction to go 3.Because the polymerase can only add a nucleotide at a boundary between double-stranded and single-stranded DNA 4.Because the two strands of genomic DNA would re-anneal without the primers
3.Because the polymerase can only add a nucleotide at a boundary between double-stranded and single-stranded DNA
If your probe is complementary to the flanking sequence of this minisatellite, how many alleles (bands) should you expect to see? 1.1 2.2 3.Either 1 or 2 Many
3.Either 1 or 2
An expert in forensic DNA statistics testifies that the defendant matches the DNA profile from a crime-scene bloodstain, and that the probability of a random match is only 1 in one million. For which of the following probability statements is "1 in a million" the correct answer? 1.Prob(Guilty | DNA match) 2.Prob(DNA match | the defendant) 3.Prob(DNA match | random person) 4.Prob(Innocent | DNA match)
3.Prob(DNA match | random person)
At trial, a forensic DNA analyst testifies that the defendant's DNA profile exactly matches the profile obtained from a blood stain at the crime scene. She also testifies that the random probability of such a match is 1 in 10 million.The prosecuting attorney argues to the jury that the DNA evidence is so overwhelming that they must vote to convict. 1.This is a reasonable argument. 2.This argument is flawed, because the defense has not yet been allowed to address the jury. 3.This argument is flawed, because the low probability of a random match does not itself mean a high probability of guilt. 4.This argument would be improved if the prosecuting attorney were to provide DNA tests for a large number of randomly chosen individuals.
3.This argument is flawed, because the low probability of a random match does not itself mean a high probability of guilt.
Which of the following is NOT a phase of a PCR cycle? 1.Denaturation. 2.Annealing. 3.Translation. 4.Extension.
3.Translation.
The actual, specific, role of DNA forensic analysis in a criminal case is to provide evidence that will 1.support determination of the guilt or innocence of the defendant. 2.determine the identity of the source of a biological specimen. 3.determine the probability that a person chosen at random would have the same genotype as that of a biological specimen. 4.determine the DNA sequence of a biological specimen.
3.determine the probability that a person chosen at random would have the same genotype as that of a biological specimen.
If D = observed data and H = a hypothesis, when we write:Pr(D|H)it means the 1.probability that the hypothesis is true . 2.probability that the data were correctly observed. 3.probability that the specified data would be produced, given that the hypothesis is true. 4.probability that the hypothesis is true, given that some specific data were collected.
3.probability that the specified data would be produced, given that the hypothesis is true.
Which of the following would be the best genetic marker for forensics? 1.A marker where every individual has a unique allele. 2.A marker where every individual has one of two alleles. 3.A single-nucleotide DNA marker. 4.A marker where there are many different alleles present in the population.
4. A marker where there are many different alleles present in the population.
The human genome is composed of 1.genes, mostly. 2.genes, plus some stuff in between the genes that mostly controls gene expression. 3.genes only make up a small fraction of the genome - everything else is just random sequence. 4.genes only make up a small fraction of the genome - and there are a lot of different kinds of things in the non-gene regions.
4. genes only make up a small fraction of the genome - and there are a lot of different kinds of things in the non-gene regions.
Which of the following is NOT a required component of a PCR reaction? 1.Primers 2.DNA polymerase 3.Deoxynucleotidetriphosphates 4.ATP
4.ATP
At 94°C 1.primers cannot anneal to genomic DNA. 2.genomic DNA becomes single-stranded. 3.most proteins are denatured (their activity is destroyed). 4.proteins from microorganisms that live in hot springs are not denatured. 5.All of the above.
5.All of the above.
A = 0.7 a = 0.3 What is the genotype frequency of AA? 1.0.14 2.0.49 3.0.049 4.0.42 5.Not enough information is provided to determine the genotype frequency
5.Not enough information is provided to determine the genotype frequency
Explain how PCR primer locations, STR flanking sequences and STR alleles together determine amplicon size
50 + 100 is flanking sequence =200 7 repeats x 4 b/c tetranucleotide 200 + (7x4) = 228 bp
Which conditional probability corresponds to the 1-in-73,000,000 number presented at trial? (A)Pr(E|I) = probability that the evidence would have been observed, given that the suspect is innocent (B) Pr(I|E) = probability that the accused is innocent, given that the rare event has occurred 1.(A)only 2.(B)only 3.Both (A)and (B) 4.Neither
A only
Explain the cause and effects of primer-dimers in PCR
A primer-dimer is a PCR by-product formed by primers attaching to each other the cause is to low of annealing temp. during PCR, contamination of template, a lot of primers the effect of primer-dimers is that it can kill the target
Explain the naming convention for alleles at an STR locus
D#Sxxxx # - chromosome number S - STR locus xxx - just a consecutive number
the purpose and use of "DNA ladders"
DNA ladder is size standard : fragments we know how many bp large is each band purpose is to identify the size of unknown DNA
Explain and distinguish between the Prosecutor's Fallacy and the Defense Attorney's Fallacy--Defense Fallacy
Defense Fallacy infers that if the probability of a suspect is 1/y then x/y people could have committed the crime. Ex. A town of 1,000,000 people have random match probability(RMP) of 1/10,000. The defense attorney may apply that this suggests that any one of 100 people in the city might be the culprit. This fallacy underemphasizes the weight of DNA evidence and causes jurors to ignore other evidence by focusing on what they perceive to be important figure.
Where does the energy for DNA polymerase come from? (the most proximate source - so not "the Sun") A.ATP B.GTP C.Electron transport D.Photosynthesis E.None of the above
E.None of the above
Describe the organizational structure of the CODIS system
Goes from local--- state--national Only the state DNA Identification Systems can communicate with the National DNA Identification System
Diagram and explain modern "hot-start" PCR
Helps prevent non-specific amplification
Interpret an electropherogram/chromatogram
If the detector is in a fixed position, the smaller fragment will arrive at the detector first the size of PCR product = size of both flanking region + size of the repeat unit
For a particular RFLP locus, design both a single-locus and a multi-locus probe
If the probe is complementary to the repeat units we have multi locus probe if probe is complementary to to the flanking sequence(single copy DNA) we have single locus probe
Distinguish between a genetic marker and a "gene" or a "locus"
Locus is simply the location on a chromosome where specific gene is found. Gene is sequence of nucleotide in DNA
Describe what information is not associated with a CODIS DNA profile
No names or other personal identifiers of the offenders, arrestees, or detainees are stored using the CODIS software for both in target and candidate columns
Describe how to move an STR locus using a mobility modifier
Non-nucleotide linkers are synthesized into the primer between the fluorescent dye and 5'end of the primer sequence. During PCR amplification, the dye and linker are incorporated into the amplicon. With the added non-nucleotide linker, the mobility of the generated STR allele will be shifted to a larger apparent size during electrophoresis. This shift of STR alleles for a particular locus then enables optimal inter-locus spacing for STR loci labeled with the same fluorescent dye without having to alter the PCR primer binding positions
List and describe the separate database components of the NDIS
Offender database(Index)-Contains DNA profiles of individuals convicted of murder and felony sex offenses Forensic Index-Holds profiles developed from crime-scene or other evidence in an investigation -Identity of the source is unknown Arrestee Index •Holds DNA profiles of individuals arrested -Under various restrictions
Discuss the challenges associated with multiplex PCR of STR loci
PCR products from different loci must be distinguishable All primer pair must be compatible Annealing Temp. Mg++ concentration
Explain and distinguish between the Prosecutor's Fallacy and the Defense Attorney's Fallacy---Prosecutor Fallacy
Prosecutor Fallacy is when the context in which the accused has been brought to court is falsely assumed to be irrelevant when weighing the evidence. Ex. An analyst may testify that theres " 1/x chance that someone other than the suspect left the sample" This statement ignores other crime scene evidence and overemphasizes the weight of evidence under discussion.
Define a Short Tandem Repeat locus
STR=microsatellite 2-6bp •Population genetics - usually called microsatellites
What kind of mutation at the restriction enzyme siteis probably the most common?
Single-nucleotide mutations.
Which of the following attributes should the probe possess?
Single-stranded Reverse complement to the target
Describe the difference in size resolution between agarose vs polyacrylamide gels
Southern blotting normally uses agarose gel - not enough resolution for STR alleles Polyacrylamide gel - much better resolution: able to detect differences of just 1 base pair in size
Describe what information is associated with a CODIS DNA profile
The DNA profile for the 13 core CODIS loci The Agency Identifier of the agency submitting the DNA profile; The Specimen Identification Number—generally a number assigned sequentially at the time of sample collection. The database (Index) in which the profile is stored
Explain what the bands represent in a multi-locus RFLP autoradiograph
The core (repeat) sequence of many minisatellites occurs in several different places in the genome looking at all of the places
Describe the kinds of loci that were used in forensic applications of allele-specific PCR
The most informative locus (DQa) had multiple alleles used STR loci •Use a protein-coding gene at which we already know there are multiple alleles in the population •Find the place where the alleles have a different DNA sequence •Make allele-specific oligonucleotide probes -Each probe will hybridize only to its exact matching allele •Under the right conditions •Perform PCR amplification of the region that includes the polymorphic site •Try to hybridize the PCR product to the probe •Detect if hybridization occurred
Explain what the bands represent in an in a single-locus RFLP autoradiograph
The unique / flanking sequence occurs at only one place in the genome we are looking at only one of the places where the minisatellite repeat occurs
Define and diagram a VNTR DNA locus
VNTR=Variable Number of Tandem Repeats Tandem means arranged in a line •Minisatellite= one kind of VNTR -Basic repeating unit usually 6-60bp -RFLP = Restriction Fragment Length Polymorphism •Microsatellite= another kind of VNTR -Basic repeating unit usually 2-6bp
Diagram the process of unequal exchange in a VNTR locus and describe how it can lead to the generation of new alleles
What things happen during meiosis? 1. Homologous chromosomes pair What happens when homologous parts of a homologous pair cannot line up exactly? The single copy flanking regions will pair up Some copies of the repeat will loop out
Discuss and illustrate how to distinguish between products from different primer pairs in a multiplex PCR electropherogram/chromatogram
What we detect is the size of the PCR product, not the size of the allele The size of the PCR product = size of both flanking regions + size of the repeat unit
Know when it is and is not possible to calculate expected genotype frequencies when given allele frequencies
You can always calculate allele frequencies if you know genotype frequency You can't calculate genotype frequencies from allele frequencies
Define a "genetic marker"
anything we can find in the genome that might help us distinguish one person from another is gene/DNA sequence w/ a known location on a chromosome that can be used to identify individual
Define "Minisatellite" and "RFLP" loci
basic repeating not of 6-60bp minisatellite markers called Restriction Fragment Length Polymorphism (RFLP)-difference between 2 alleles is the distance between 2 restriction enzyme
Explain why RFLP data were considered more informative than allele-specific PCR data
because it was the golden standard and required a lot of blood allele-specific PCR could only provide information about a few loci, each with only a small number of alleles.
Describe how to move an STR locus by changing the PCR primers
by making a new primer
define mobility modifier
can change the apparent size of an ampliconwithout moving the primer location
Describe and use conditional probabilities
conditional probability is the probability of one even occurring with some relationship to one or more events Formula: P(B/A)= P(A and B) / P (A)
Describe the legal basis for the CODIS system
establish a national identification index of DNA records
In the expression Pr(E|X), the symbol '|' means
given.
List the characteristics that make a genetic marker useful for human identification
having a lot of variation "highly polymorphic" it maximize the chance that 2 individuals will have different genotype
Define amplicon
is a piece of DNA or RNA that is the source and/or product of natural or artificial amplification or replication events
Define "allelic ladder" as used with multiplex STR PCR
is an artificially constructed mixture, understand how multiplex STR PCR kits work
Define "Reference Sample"
is known source taken directly from the person
Explain what it means to "move" an STR locus in a multiplex PCR panel
means to change changing the distance between the primers and repeat
A DNA chain can grow
only from 5' to 3'.
Given allele frequencies for any number of alleles at a locus, calculate the Hardy-Weinberg expected genotype frequencies
p=the frequency of dominant allele (A) q= frequency of recessive allele (a) {1 or 2 alleles} p+q=1 p^2= frequency of AA(homozygous dominant) 2pq= frequency of Aa (heterozygous) q^2= frequency of aa (dominate recessive) p^2+2pq+q^2=1 {1 or 2 alleles} •p+q+r+s= 1 {multiple alleles}
The "flanking regions" of an RFLP / minisatellitelocus are
random single-copy sequence unrelated to the repeat unit
Given a set of genotype frequencies, calculate allele frequencies
take 1/2 heterozygote take all of homozygote Add them together
If you want to visualize only one locus where this microsatellite repeat sequence is located inthe genome,
the probe should be complementary to the flanking sequence.
If you want to find all of the loci where this microsatellite repeat sequence is located in the genome,
the probe should be complementary to the repeat region.
Given the way in which two VNTR alleles pair at meiosis, determine the alleles that will result from a recombination event
unequal exchange- Mutation of minisatellite alleles
What does VNTR stand for?
variable number of tandem repeats
Discuss the advantages associated with multiplex PCR of STR loci
you can mix more than one primer pairs for PCR to work fine increases power of discrimination saves time reduce labors allows much more information to be gained from same sample
Describe the basic CODIS workflow
•A forensic laboratory receives evidence in a criminal investigation performs DNA testing. = "forensic unknown" •If you have a suspect, compare the genotype to the suspect's reference sample. If no suspect •The laboratory enters the forensic unknown profile into CODIS. -For a local case, the profile is entered into the local CODIS system and uploaded to the state level. -At the state level, the profile is compared with all the offenders from that state's database. The forensic unknown may or may not match with other DNA records at the state level •NDIS compares the profile against all 50 states' offender profiles -if there is a match, the CODIS software automatically returns messages in the system to the laboratories involved. -The local labs evaluate the matches and release that information to the law enforcement agency.
Define a casework sample, distinguish between a reference sample and a casework sample
•A reference sampleis taken directly from an individual-Identity of the source is known with certainty -Generally something recovered during an investigation •Identity of source not known
Illustrate the different kinds of STR repeats (dinucleotide, trinucleotide, etc.)
•Dinucleotide (CA)(CA)(CA) •Trinucleotide (GCC) (GCC) (GCC) •Tetranucleotide (AATG) (AATG) (AATG) •Pentanucleotide (AGAAA) (AGAAA) Hexanucleotide (AGTACA) (AGTACA)
Explain the differences between describing results of human identity testing as: exclusion / inclusion / match
•Exclusion is a definitive statement •Match ≠ positive identification is not excluded
Interpret an RFLP autoradiogram as used in forensics in terms of match vs exclusion the source
•Match: -all bands (or alleles) detectable in the evidence sample match those in a reference sample •Significance of a match depends on the probability that some other person, chosen at random, would also match the evidence sample •Not excluded: -Basically the same as 'match', but often preferred because it is less of a loaded term •Can also allow for some bands to be missing if there is a chancethey could be there but not detected •Exclusion: -One or more bands (or alleles) are present in the evidence sample but not in the reference sample •Exclusion is definitive - the person who gave the reference sample is excluded as being the source of the evidence sample
Discuss the advantages of capillary gels for analysis of DNA fragment sizes
•Size standard runs in the same capillary -No need to interpolate -No need to worry about the alignment of a lane on a slab gel •Can be highly automated •Large surface-to-volume ratio dissipates a lot of heat -Can apply much higher voltage •Shorter run times
Describe the design parameters for multiplex PCR primers, and how to determine a primer's optimal annealing temperature
•There are automated programs for primer design -Can get Tm in the right general ballpark -Very good at avoiding primer-dimers • •But: multiplex STR primer design involves many serious consraints -the need to put primers very close to the STR •To keep amplicons small -the need to have amplicons of very specific size - •Development of new primers requires months (at least) of trial and error
Describe specific properties of a genetic marker that would make it a good choice for forensic analyses
•Two parameters affect the level of variation for a locus -the number of alleles in the population -the frequencies of the alleles •(population genetics)
