bala 3

kary mullis

invented PCR (polymerase chain reaction)

francis collins

started the Human Genome Project

introns vs exons

introns

• non coding regions

• ~30,000 genes in humans

exons

• coding regions

• 5% of human genomic DNA coding regions

In a 310 capillary electrophoresis system, the amplified fragments are separated by ________ and ____________

size color

In the ABI identifiler multiplex kit, the fluorescent fragments are generated by __________________ ____________________

labeled primers

In the Identifiler STR analysis kit, that amplifies 15 STR markers and one amelogenin marker, how many primers are included?

32

Non human DNA will not interfere with forensic STR marker amplification because:

they are human-specific

The number of loci recommended for forensic human identification is __________.

20

Different electrophoretic systems.

• agarose

• acrylamide

• capillary

What is capillary electrophoresis?

rapid charged molecule separator (proteins, DNA fragments)

What are the two advantages of ABI 310 capillary genetic analyzer?

automated gel pouring

automated sample injection

The samples in the 310 capillary instrument run at 100°C during electrophoresis. True or false

false

What are the three steps in a PCR cycling and associated temperatures?

denaturation ~ 95*C

annealing of primers ~ 50-65*C

extension of primers ~ 72*C

In a PCR reaction using the multiplex human identification kit, the entire human genome is copied rather than small segments of the human DNA. True or False

false

The Promega powerplex 16 human identification kit contains__________ (#) STR markers and __________ (#) amelogenin marker.

15 1

For a 16 marker autosomal STR analysis, how many alleles are amplified?

32

What are the four different types of detection systems for the amplified STR alleles?

• capillary electrophoresis

• gel staining

• flatbed scanning

• fluorescent dye

Name any ten STR markers/loci used for forensic DNA analysis.

FGA

TH01

TPOX

vWA

CSF1PO

D3S1358

D5S818

D7S820

D8S1179

D13S317

D16S539

D18S51

D21S11

What are the precautions that one will employ to avoid contamination in a forensic DNA analysis laboratory.

• separate pre and post PCR areas

• pipette sets for each area

• disposable gloves

• laminar flow hood

• aerosol resistant tips

• prep are free of DNA

• UV radiation and 10% bleach cleaning

What are the advantages of PCR technology in forensic DNA analysis?

• small amount of DNA

• degraded DNA

• multiplex saves time and sample

• no non human DNA interfence

• easy set up with kits

Describe the structure of a 310 genetic analyzer using diagram and label the parts.

In an electropherogram, you see one peak for one locus and two peaks for another locus. Why?

1 peak - homozygous

2 peaks - heterozygous

Acrylamide gel image and allele calls using an allelic ladder

compare sample

ladder bands

In a multiplex PCR amplification, the two PCR Products (alleles) of the same size from the same locus (for example, both 199 base pairs long) can be separated in a capillary electrophoretic system. True or false

color

Number of capillaries and electrodes in an ABI 310 genetic analyzer.

1 capillary

2 electrodes

In an electropherogram, the big dye blob in the beginning of the raw data is due to ____________________

unused primers

In a capillary electrophoresis the smallest fragments to reach the detection window are_____________

primers

Auto sample injections are one of the disadvantages in the 310 capillary instrument. True or false

false

Analysis parameters and other built-in parameters for sample data analysis.

range of analysis, baseline, matrix file, size standard, name of kit, number of colors

Number of alleles in autosomal versus Y STR markers.

autosomal ~ 2

Y STR ~ 1

There is a PCR multiplex kit manufactured by a private company and it is called Powerplex 35GY 8C system. What is the 8C refers to?

8 color(s)

Y chromosomal lineage.

the paternal ancestry of an individual traced through the Y chromosome

How will you identify if a sample is coming from a single source or mixed samples?

single ~ 2 or less

mixed ~ more than 2

The process of transferring DNA from an agarose gel to a nylon membrane is called ______________.

southern blotting

The process of transferring RNA from an agarose gel to a nylon membrane is called ______________.

northern blotting

The transfer of DNA from an agarose gel to a nylon membrane is associated with this person.

edwin southern

ABI 310 – instrument parts and internal organization of parts (# of electrodes, # of capillary, # of buffer vials, # of samples, Argon Ion laser, CCD camera, Inner diameter/length of capillary etc.)

diameter ~ 50μm

length ~ 47 cm

2 electrodes

1 capillary

2 buffer vials

48 - 96 samples

argon ion laser ~ excites dyes to fluoresce

CCD camera ~ calculates size/quantity of fragments

What are the two primary differences between forensic criminal case DNA profiling and paternity testing?

forensic criminal case

• 2 profiles needed ~ evidence and suspect

• all alleles have to match

paternity testing

• 3 profiles needed ~ mother, father, child

• 1 allele of child has to match father

How an allele frequency database is established?

compiling data from literature, research and studies

What are the chances of picking up an ace in a deck of 52 cards?

4/52

7%

What are the two formulas to calculate genotypic frequency for homozygous and heterozygous locus?

homozygous

dominant ~ f(AA) = p²

recessive ~ f(aa) = q²

heterozygous

f(Aa) = 2pq

What is cumulative genotypic frequency?

combined proportion of genotypes within a population

p² + 2pq +q² = 1

What is the reason to calculate the Random Match Probability of a DNA profile for all three racial groups in US?

to ensure that the significance of a match is not biased by unknown ancestry

If there are three alleles present in a locus and mixture is ruled out, what will be your conclusion regarding that locus?

it is tri-allelic

How do you differentiate a mixture from a Tri-allelic pattern.

mixture

• more than 2 alleles on multiple loci

• peak heights unbalanced

• 3 allele pattern not present in other samples

tri-allele

• alleles on 1 locus

• peak heights are equal

• 3 allele pattern present

Draw the two possibilities to show a tri-allelic pattern

2 peaks equal 1 peak

3 peaks are equal

Calculation of random match probabilities.

heterozygous ~ 2pq

homozygous ~ p²

What is a product rule?

formula to find the derivative

The genotypic frequency is calculated from ……………..

allele frequencies

The sequence of events in a criminal DNA profiling in chronological order:

1. collection

2. extraction

3. quantification

4. amplification

5. analysis and interpretation

6. comparison and reporting

7. database comparison

What are the two caveats that are added in the DNA report when issuing an identity statement (that the suspect is the source of DNA in the evidence material)?

1. absence of an identical twin

2. to a reasonable degree of scientific certainty

The tri-allelic pattern in an STR profile refers to __________

the presence of 3 alleles

The nucleotide that is added at the 3’ end of PCR products in a PCR reaction is __________ (non- template dependent addition).

adenylation

Define adenylation.

AMP molecule attaches to proteins amino acid

The mutation at the 3’most primer binding site will lead to:

a. No issues

b. Poor amplification

c. No amplification

d. None of the above

c. no amplification

The stutter products are normally ________ bases smaller than the real allele, it is associated with.

4

Stutter products are generally __________ % or smaller than the height of the allele causing it.

10

Stutter products are generated during the PCR process. Yes/no

yes

Stutter products are avoidable during the PCR reaction. Yes/no

no

If the stutter percent in a locus is above the normal stutter % level, what will be your conclusion?

there is a mixture of DNA from more than 1 source

What are proof reading Taq DNA polymerase enzymes?

PCR enzymes to detect and correct mismatched bases

In a microvariant, the .3 in the 9.3 THO1 locus refers to_________

3 base pairs

Give an example of a microvariant repeat (STR repeat pattern with diagram) and the resulting allele call.

TH01

9 full AATG repeats and 1 partial repeat of ATG

call = 9.3

Null alleles refer to the _______________ of an allele in a locus.

non-amplification

Mutations can happen in the primer binding sites. True/false

true

What will be the effect of the primer binding site mutation at the 5’most end of the primer?

a. No amplification

b. Poor amplification

c. Mostly, no effect

d. None of the above

c. mostly, no effect

What will be the effect of a mutation in the middle of a primer binding site.

• reduced efficiency

• lower yield

Name any five information present in a Forensic DNA analysis report.

1. case details

2. result details

3. final interpretations

4. signatures

5. type of DNA

What is an identity statement?

a formal conclusion reached by forensic scientist