Advanced Cell Bio Lab Midterm

C. Elegans is multicellular/unicellular prokaryote/eukaryote

multicellular eukaryote

How do we precipitate BLT beads with bound DNA at the bottom of the PCR tube without centrifugation?

Magnetic stand for magnetic bead

Which two additional adaptor types are added to DNA fragments during the BLT PCR procedure?

Flow cell complement and index

During the tagmentation procedure DNA is

simultaneously fragmented and tagged with adapters on both ends of the fragments that are primer binding sites for the indexing primers (transposomes do this)

During tagmentation, your DNA, along with the newly attached sequence tags

remains permanently bound to the tagmentation beads

What does Cq represent?

The cycle at which fluorescence surpasses the detection threshold

What is being measured at A280?

protein concentration for contamination

Why is E. coli strain OP50 used for feeding C. elegans versus other common lab strains of E. coli?

This specific strain was chosen because it is a uracil auxotroph, meaning it requires uracil for growth but cannot synthesize it on its own. This characteristic helps prevent overgrowth and the formation of a thick bacterial lawn on the agar plates, ensuring clear visibility of C. elegans during the experiment. The E. coli OP50 strain included the pBAD plasmid, which provides antibiotic resistance. This allowed the bacteria to grow on antibiotic-containing agar plates while preventing contamination from other bacterial strains. By maintaining a controlled bacterial lawn and reducing external contamination, this setup ensured optimal conditions for observing C. elegans behavior in the chemotaxis assay.

Spectrophotometry advantages:

we can determine purity of nucleic acids (like ssDNA, dsDNA, and RNA)

Spectrophotometry disadvantages:

cannot tell us about dsDNA specifically, less sensitive, cannot distinguish RNA from DNA

Qubit advantages:

highly sensitive and specific to dsDNA only

Qubit disadvantages:

cannot measure purity and can only read dsDNA

Purity formula

(A260 reading – A320 reading) ÷

(A280 reading – A320 reading

Final DNA concentration formula

Concentration (μg/ml) = (A260 reading – A320 reading) × dilution factor × 50μg/ml

Good quality purity

between 1.8-2

1.8 purity reading

pure DNA

greater than or equal to 2.0 purity reading

high RNA concentration or denatured DNA

Less than or equal to 1.6 reading

high protein contamination or other contaminations

DNA/RNA absorbs

260 nm

A320 reads

turbidity and light scatter (any contaminants that cause light scatter)

Life-span of C. elegans

2-3 weeks, L1-L4 juvenile stages

Primitive behaviors of C. elegans

feed, reproduce, locomotion

Complex behaviors of C. elegans

learning, mating, social

Daf-18 gene has a human homologue:

PTEN gene (also encodes for a phosphatase)

Wild-type C. elegans should know NaCl =

food

Inhibitor remover solution

neutralization buffer that contains potassium acetate, removes inorganic and organic contaminants, and causes SDS to precipitate out of the solution

PowerBead solution has

buffer and salts to disperse microbial cell solution before lysis

Neutralization buffer is added

after the lysis procedure

SB solution (DNA binding solution)

high guanidine salt solution for DNA-spin column binding

CB solution

ethanol-based for washing column-bound DNA (contains guanidine salts and ethanol), ethanol allows the precipitative state of DNA by further decreasing solubility

Elution buffer (or qH20) (or any low ionic buffer)

washes off bound DNA, elutes the DNA

Lysis buffer

contains SDS which breaks down FAs and lipids, denatures proteins and gives it a negative charge

Bead suspension buffer

has salts to disperse cell solution before lysis

how does guanidine salt decrease solubility?

the positively charged guanidine salts bind to the negatively charged silica membrane, helps to disrupt hydration shell around DNA, making it less soluble

How does DNA bind to the silica membrane?

Both are negatively charged, but an ion bridge is created to allow attachment due to the guanidine salts

How do strong chaotropic salts (such as guanidine) modify the structure of water?

Changes how water interacts with moieties in the solution, forces nucleic acids to interact with silica and compete with water

Why do we use the strain of bacteria (OP-50)?

requires uracil to grow, contains the pBAD plasmid, gives E. coli antibiotic resistance, which allows it to avoid other bacteria contamination on the plates

C. elegans hermaphrodites

contain both male and female sex organs, capable of self-fertilizing

C. elegans male

contain only male organs, cannot self-fertilize, but can inseminate a hermaphrodite, distinguished from hermaphrodites by the presence of a specialized tail used during mating

when crowded or without enough food, L1 and L2 C. elegans can enter an alternative specialized larval stage called

dauer (and cannot feed due to oral orifice being plugged)

deletion/mutation of daf-18 gene causes

defect in associative learning behavior (cannot make association between a good feeding environment and salt), migrate randomly across the plate, can still sense NaCl but cannot learn

positive chemotaxis/negative chemotaxis

movement towards chemical/movement away from chemical

chemotaxis index formula

(# C. elegans on NaCl side – # C. elegans on control side) /

(# C. elegans on NaCl side + # C. elegans on control side

chemotaxi index = 0

same number of worms moved toward the chemical as moved away from the chemical

The more damages there are to the DNA, the (more/less) cycles needed to reach threshold value

more

UV light exposure primarily causes

ss-DNA breaks

Ionizing radiation primarily causes

ds-DNA breaks

pyrimidine dimers

two neighboring nucleotdies bind to each other and causes the molecule to stretch and bend which can block DNA polymerase leading to replication errors (form cyclobutane ring structures)

ROS can sometimes

oxidize guanine and result in a guanine-like nitrogen base that can no longer pair with cytosine

How do strand breaks occur?

when the bonds between the phosphate group and the sugar group of two neighboring nucleotides disconnect

When DNA damage is detected the cell can respond by either:

reversing the change, replacing a small section of DNA, freezing the cell so it can no longer divide (senescence), or apoptosis

Samples that have a high proportion of damaged DNA have a lower rate of

amplication and less amplified products than less damaged DNA samples

Quantitative PCR

allows differences in DNA damage between samples to, not only be identified, but to also be precisely described

in qPCR DNA amplification is observed and measured

during the experiment rather than following the experiment

how does qPCR work?

performed in specialized thermal cyclers that have been equipped with lasers (to excite the fluorescent molecules) and detectors (to measure and record the signal that dyes or probes produce during amplication) to be used to calculate the starting amount of non-damaged DNA in a sample

The increase in amplified DNA during qPCR related to an

increase in the fluorescent signal

when is the best time to describe the relationship between DNA amount and a fluorescent signal?

after the signal has exceeded the background noise but before it begins to plateau

DNAse I

an enzyme that helps with cleanup following cell apoptosis by causing dsDNA breaks

how can the relationship between signal and DNA concentration be described?

through creation of a standard curve

Can we just sequence isolated DNA?

No because it has to be fragmented and “tagged” with adaptors

How does tagmentation work?

DNA fragments bind to beads’ transposomes, DNA is ligated with adapters, fragments are now released (only with adaptor and only above a certain size)

why are more fragments the same length?

the distance between transposomes is fixed

Reduced cycle amplification adds

an index and also adds sites complimentary to flow cell sequencers

when are the first adaptors added

during bead-linked tagmentation (first adaptors - reading sequencing primer)

Second and third adaptors are added during

Reduced-cycle amplification (second and third adaptors - Indexes and flow cell complement)

How does the sequencer know whose sample is where?

each sample is tagged with a unique i7 and i5 adapter (serve as “barcodes” or “tags”)

DNA photolyase

uses visible light in order to break cyclobutane rings around the pyrimidine dimers

What do the sequence tags include?

primer binding sites that indexes are gonna bind to (complement to tags), unique index sequences, and flow cell complement

Tagmentation of genomic DNA step

DNA is fragmented and tagged with adapter sequences using BLT (prepares DNA for sequencing)

Post-tagmentation cleanup step

unwanted byproducts and excess reagents from tagmentation are removed using a wash buffer

Amplify tagmented DNA

PCR step to add index adapters and complete library constructure

Clean up libraries step

DNA libraries purified using a bead-based method to remove contaminants and DNA, of the right fragment size, was released from the beads using low ionic strength

Flow cell binding sequence

allows the sequencing library to bind to the flow cell and prevents libraries from being washed away during the sequencing process

Sequencing primer binding site

primer binds to this region during sequencing and initiates the sequencing by synthesis of target DNA