Competency Test (MASTER SET)

What is a mitochondrion?

an organelle in the cytoplasm of eukaryotic cells whos primary function is ATP synthesis

the mitochondrion creates ATP through a process known as ___________________

oxidative phosphorylation

What is the purpose of the mitochondrion's outer membrane?

1) communication with other orgnaelles

2) maintains shape

What is the purpose of the mitochondrion's inner membrane?

1) oxidative phosphorylation

2) communcation with nearby mitochondria

Which of the mitochondria's membranes contains highly folded cristae which increase its surface area?

the inner membrane

Where is mtDNA located?

in the matrix of the mitochondria

T/F: there is at least one copy of mtDNA per mitochondria

true

Why is mitochondrial DNA more useful for limited or degraded biological material over nuclear DNA?

1) hundreads to thousands of copies per cell

2) circular nature makes it less suspectible to exonucleases

3) necapsulation (2 walls) increases its survival rate over time

T/F: mtDNA undergoes recombination and has replication repaire mechanisms

false

The mutation rate in the non-coding regions is at least ________________________________________ than that of nuclear DNA

5-10 times higher

What allows for sequence variability between unrelated individuals in their mtDNA?

the high mutation rate in non-coding regions

How is mtDNA inherited?

maternally

T/F: mtDNA is haploid, meaning there is only one allele for each loci

true

T/F: mtDNA is NOT unique to individuals. Rather, since it is inherited maternally, everyone in that maternal line will share the same mtDNA

true

Why is mtDNA inherited maternally?

at conception, only the sperm's nucleus enters the egg and joins directly with the egg's nucleus. The egg contains mitochondria which are passed to the zygote.

What statistic is used to calculate the likelihood of a mtDNA haplotype

the counting method

What is the counting method?

a statistical calculation that determines the number of times the sequence has been seen in the population size

What is the main weakness of the counting method?

it is dependent on the number of samples in the database

What types of cases is mtDNA useful in?

1) cases with no direct references

2) missing persons

3) mass disasters

What are the characteristics of mtDNA?

1) circular

2) doubel stranded helix

3) light and heavy strand

4) two main regions (coding/non-coding)

What is the heavy strand of mtDNA?

the strand with the higher number of guanine residues and therefore the heavier molecular weight

What is the light strand of mtDNA?

the strand that is cytosine/thymine rich

What is the coding region of mtDNA?

the area that contains the genetic information for energy production, signaling, growth, and apoptosis (approx 37 genes)

T/F: the coding region is used for mtDNA testing

false!!!

What are the two exceptions for mtDNA testing in the coding regions?

1) 12S rRNA region for species ID

2) Next Generation Sequencing --> sequences the whole genome

What are the other two names for the non-coding region of mtDNA?

1) Displacement loop (D-loop)

2) Control Region (CR)

Where is the non-coding region of mtDNA located?

it begins at the heavy strand origin of replication and is about 1122 base pairs long

what is the non-coding region of mtDNA?

the area of the mitochondrial genome that does NOT code for any gene products and is hihgly polymorphic

Where are the hypervariable and variable regions located?

in the non-coding region of the mtDNA

What is DNA and what does it stand for?

deoxyribonucleic acid, it is the biomolecule that carries the genetic code for growth, function, and reproduction in all living organisms

Where does DNA reside in a eukaryotic cell?

nucleus

mitochondria

What are the names of the four nitrogenous bases that can make up a nucleotide?

guanine

cytosine

adenine

thymine

Which nitrogenous bases bond together?

Adenine with Thymine

Cytosine with Guanine

The nitrogenous bases can be classified as either pyrimidine or purine. Which ones belong to which classification ?

pyrimidine - thymine and cytosine

purine - adenine and guanine

What is the main difference between a pyrimidine and a purine?

Purines have a double-ring structure (one 6 one 5)

Pyrimidines have a single-ring structure ( 6 membered)

In addition to the nitrogenous bases, what are the other two components that make up a nucleotide? Where are they located in relation to the nitrogenous base?

Pentose Sugar: Sits in the middle. nitrogenous base sits are carbon 1

Phosphate Group: Sits on the outside/edge. It connects to the pentose sugar at its 5′ carbon.

An organism that has the same two copies of a gene or allele is called ___________

homozygous

T/F: nuclear DNA is in the conformation of a double helix. Mitochondrial DNA is also in this conformation

true

T/F: there are more copies of nuclear DNA in a cell than mitochondrial DNA

false

What portion of the mtDNA genome is used in Sanger sequencing and why?

control region (D loop) because it doesn't code for genes which allows for more variability and thus differences between maternal lines for identification

How does the Bioanalyzer separate DNA strands?

1) DNA is loaded into a chip that has microchannels filled with a conductive gel matrix.

2) An electric field is applied

3) DNA molecules move towards the positive end because they are negatively charged

4) The gel acts as a sieve separating everything by size and charge

How is the DNA detected in the Bioanalyzer to create the electropherogram?

1) DNA is mixed with an intercalating dye.

2) After the DNA passes the detection point and is hit with a laser

3) This causes the dye to fluoresces which is captured by a camera

4) That signal is then converted into an electropherogram

What are the four components that are included in the bioanalyzer kit?

Dye Concentrate

Gel Matrix

Marker

Ladder

What are the three types of assays used for the BA?

7500, 12000, and High Sensitivity

The BA quantifies DNA using on chip ______________________

Gel electrophoresis

What are the Pros to doing on chip gel electrophoresis?

Decreases separation time

reduces sample and reagent consumption

What two things are the microchannels of the BA chips filled with?

A polymer and fluorescent dye

What two reagents are combined to create the BA's Gel Dye Mix?

The Gel Matrix and the Dye Concentrate

What is the purpose of the Gel Matrix reagent for the BA?

This is the polymer that is pumped through the channels on the chip to help sieve the DNA fragments

What is the purpose of the Dye Concentrate reagent for the BA?

This is the fluorescent dye that intercalates with the DNA and allows for detection of fragments

What is the purpose of the ladder with the BA reagents?

Helps to determine size and concentration of DNA that is detected

What is the purpose of the marker with the BA reagents?

Helps to determine size and concentration of DNA that is detected

What is the validated concentration range for the BA's 7500 assay?

Down to 3.125ng/uL

What is the validated concentration range for the BA's HS assay?

195pg/uL to 6.25 ng/uL

What is the validated concentration range for the BA's 1200 assay?

0.87ng/uL to 42.37 ng/uL

What is the recommended use for the BA's HS assay?

For extract quant

NEB Next Post Library

Nextera Pools

What is the recommended use for the BA's 7500 assay?

Capture pools

KAPA HyperPrep and SNP

KAPA Hyperprep Post Library

What is the recommended use for the BA's 12000 assay?

Nextera LR amplicons

What is the size range (bp) that the BA's HS assay designed to detect?

50-7000 bp

What is the size range (bp) that the BA's 7500 assay designed to detect?

100-7500 bp

What is the size range (bp) that the BA's 12000 assay designed to detect?

100-12000 bp

How is the standard curve of the BA generated?

A plot of the ladder base pair peak sizes versus migration time is created

What are the two ways that the BA can show the results of a run?

As an electropherogram and as a gel

What is the purpose of internal standards used on the BA?

Used to align the ladder data with sample data and compensate for drift effects that could occur over the course of a run

What should you expect to see on the electropherogram for the HyperPrep SNP Pool?

A lower marker peak at 50bp

An adapter dimer peak between 100 and 170bp

A DNA smear with most fragments falling between 170 and 400 bp

An upper marker peak called at 10380bp

What should you expect to see on the electropherogram for the HyperPrep Global Mito Pool?

A lower marker peak at 50bp

An adapter dimer peak between 100 and 170bp

A DNA smear with most fragments falling between 170 and 400 bp

An upper marker peak called at 10380bp

If the concentration for the full-size range with the 7500 is greater than 50ng/ul what should you do?

Dilute the pool to less than 50ng/ul with TE and re-quantify it

If a pool quants below the validated range for the 7500 assay, what do we do?

We re-run with the High Sensitivity (HS) assay

What does quantification with the BioAnalyzer tell us?

The molarity of the pool (nM) which is used for sequencing

Bioanalyzer reagents should be left out __________ prior to use. Why?

30 min

they must come to room temperature prior to use

Is there ever a time when you would switch between the BA assays?

Yes, when the concentration is found to be not in the range of that assay

What is used to determine the base pair size of a peak and how does the software do this?

the migration time of unknown DNA fragments is compared to the standard curve generated by known DNA fragments from the ladder.

Internal markers and assay-specific calibration help ensure accurate sizing across the assay's range.

What is run with each sample on the bioanalyzer and used to align the ladder data to the sample data?

internal lane standards

an amplicon must exceed _______ to be quanted

20 FU

the average molarity of a library pool is 25,154.9 pmol/L. HOw would you convert this to nM?

move the decimal place to the left three times

What does PPE stand for?

Personal protection equipment

What is the purpose of PPE?

To reduce the risk of injury or impairment from chemical, radiological, or physical hazards when controls aren't enough

Protection from PPEis provided against these three types of hazard transfer modalities…

Absorptions, inhalation, and physical contact

What is the minimum PPE for all labs?

Safety goggles, disposable lab coat, gloves (latex or nitrile)

What are some additional PPE options available at AFDIL?

Face mask, sleeves, double gloving, cut resistant gloves for sharp instruments

What in the laboratory dress code in regards to footwear?

Close toed shoes are required with no holes. Shoes like flip flops, sandals, and other open toed shoes are not permitted

What in the laboratory dress code in regards to personal attire and accessories?

Professional clothing for lab duties, hair and beards tied back or secured, no loose or dangling jewelry

T/F: food/drinks are allowed in the lab/lab fridges

FALSE

T/F: smoking is prohibited anywhere inside the building

True

Smoking is prohibited within _________________ of any area designated as flammable chemical storage

50 feet

What should employees who handle hazardous materials do whenever they find out they are pregnant?

Notify their supervisor immediately à they will be removed from those duties and reassigned to other tasks

What is the NFPA diamond?

A standardized system developed by the national fire protection association to quickly identify chemicals for fire fighters

Who is the primary audience for the NFPA diamond?

Emergency responders for rapid assessment during a fire, spill, or other emergency

T/F: the NFPA diamond is an OSHA standard

False, but it is widely adopted standard by industry

How is the NFPA diamond divided?

Into four color sections each rated on a scaled from minimal hazard (0) to severe hazard (4)

What do the colors on the NFPA diamond mean?

Red - fire risk

blue - health risk

yellow - reactive/instability

white - special hazards

What is the Globally Harmonized System of Classification and Labeling of Chemicals?

A universal standard from the UN for classifying and labeling chemicals

What is the purpose of GHS?

The create a single, worldwide system for hazard communication, reduce confusion between different countries regulations

What is the purpose of the bloodborne pathogen exposure control SOP?

To reduce the risk of occupational transmission of infections from human blood and other potentially infection materials (PIM)

What are two examples of key pathogens that can be transmitted through contact with infected blood or tissue?

Hepatitis B and Human immunodeficiency virus (HIV)

What are some potentially infectious materials that come from human bodies?

Semen, vaginal secretions, CSF, pleural/pericardial/peritoneal fluid, saliva, tissue/organs/bone, any body fluid visibly contaminated with blood

Where are the bloodborne pathogen spill kits normally located?

In every lab by the sink

How should flammable liquids be stored?

In the NFPA approved flammable storage cabinet labeled flammable

Where are chemical spill kits located?

In every lab near the sink

What should you do if an accident occurs AND item from the first aid kit is used?

Document it on the first aid kit log sheet and notify appropriate personnel