Molecular Diagnostics Chapter 4

gel electrophoresis

Procedure used to separate and analyze DNA fragments by placing a mixture of DNA fragments at one end of a porous gel and applying an electrical voltage to the gel

Is the anode positive or negative? What about the cathode?

Positive, negative

DNA/RNA are positively/negatively charged? They migrate towards anode or cathode?

negatively, anode

Do large or small size DNA fragments migrate faster?

small, easier to move through membrane

Heteroduplexes

A double-stranded nucleic acid containing one or more mismatched (noncomplementary) base pairs. RNA folding on itself when single stranded

agarose gel

a jelly-like slab used to separate molecules on the basis of molecular weight, polysaccharide that comes from seaweed

concentration of agarose in a gel determines the size of spaces in the gel. 3% agarose resolution is best for _______bp of DNA. 0.6% is best ________bp of DNA.

50-100 bp and 1000, 20,000

Are low concentration agarose gels easily broken?

yes

Electroendosmosis

This electrophoresis error is caused by cations in the buffer moving in the opposite direction which slows/distorts migration of samples

Pulse field gel electrophoresis (PFGE)

used to separate especially long strands of DNA by length in order to tell differences among samples. Does so by alternating current in different dimensions during migration of sample. Requires long separation times and temperature control. 50,000-250,000bp

FIGE

field-inversion electrophoresis, samples move forward and backward due to the changing current/pulse field

polyacrylamide gel

-porous matrix mixture used in electrophoresis typically for smaller fragments

-single bp resolution so can be used for sequencing and mutation analysis

-synthetic can be manipulated to have exact same conditions or necessary conditions

w/v, t, C PAGE gels

IF, T value 6% and 19:1 acrylamide:bis, then C=?

w/v total percentage concentration of acrylamide and t the percentage of monomer, T=acrylamide and cross linker, C= percentage of monomer that is a cross-linker.

C=1/20 or 5%

Can PAGE gels polymerize upon cooling?

No need catalyst such as APS and TEMED or light

Are DNA strand single or double stranded for capillary gel electrophoresis?

single, very important so that separation will occur by size

How does capillary electrophoresis resolve DNA?

by size through a polymer matrix and by charge through a negatively charged array and current

Do negatively charged small DNA fragments travel faster than large fragments through capillary electrophoresis?

yes

capillary electrophoresis

A method of separating DNA samples based on the rate of movement of each component through a gel-filled capillary while under the influence of an electric field

Is traditional electrophoresis or capillary electrophoresis faster?

capillary

Electrokinetic Injection

In capillary electrophoresis, the use of an electric field to inject sample into the capillary. Because different species have different mobilities, the injected sample does not have the same composition as the original sample. Utilizes a transient positive charge to attract DNA prior to injecting

What are the advantages of capillary electrophoresis over traditional gel electrophoresis?

Faster, no lane to lane variation as the samples are injected together, direct software program hookup for analyzing results, increased sensitivity, multiple color-detection systems

What are the disadvantages of capillary electrophoresis over traditional gel electrophoresis?

-costly to buy instrument

-requires fluorescent labeling of sample

What s the purpose of a buffer system in gel electrophoresis?

Carry the current and protect the sample

buffer

A weak acid that minimizes changes in pH when extraneous acids or bases are added to the solution. Typically the pH of a buffer is near it's pKa

equilibrium of acid and base in solution equation

Ka= [H+][OH-]/[HA]

How is Ka typically expressed?

pKa=-log[Ka]

Henderson-Hasselbalch equation

pH = pKa + log [A-]/[HA]

How does a high buffer concentration effect gel electrophoresis?

increases conductivity of electrophoresis system which results in higher temperature, this can denature DNA and decrease stability.

High buffer concentrations must be offset by ________________

low voltage

Why is Tris a good buffer?

large molecule with low charge to mass ratio so runs slowly in the buffer system

What a re examples of buffers used in DNA gel electrophoresis?

Tris, Tris borate EDTA, boric acid, TAE, TBE, TPE

What are examples of DNA denaturing agents used in gel electrophoresis?

formamide and heat -denatures DNA so long strands will migrate at similar speeds

urea-prevents reaneealing

How is RNA denatured during electrophoresis?

more challenging because secondary structure bonding is stronger compared to DNA hybridization between complementary strands, denaturant MMH, formaldehyde, or glyoxal can be used

What are examples of buffers used in RNA electrophoresis?

10mM-sodium phosphate, MOPS, EDTA

pH drift

decrease of pH at the cathode and an increase of pH at the anode

A______ connector is connected to the positive end and a ____________ connector is connected to the negative end of a gel electrophoresis setup (red/black)

red, black

When loading a sample into a gel electrophoresis well what two additional factors must be loaded?

tracking dye-not associated with the sample and notifies you to stop electrophoresis prior to sample running off the gel (monitors progress of run) and density agent which makes the sample heavier than the buffer so it does not float out of the well

Name types of tracking dyes

bromaphenol blue xylene cyanol

intercalating agents

Planar molecules that mimic base pairs and are able to slip in between the stacked nitrogen bases at the core of the DNA double helix, resulting in insertion or deletion of a single nucleotide pair. A source of induced mutation. stack between the nitrogen bases ind boule stranded nucleic acid, are mutagens example: EtBr

Types of minor groove binding fluorescent markers for gel electrophoresis visualization

SYBR green, I for ds DNA II for ss RNA/DNA,. SYBR gold

What is the advantage of SYBR green over EtBr for gel electrophoresis nucleic acid staining?

EtBr is a mutagen and is toxic, SYBR green is not

Silver staining

used to detect nucleic acids after gel electrophoresis, gel is washed with silver nitrate or silver diamine and stained the precipitated with formaldehyde

After completion of the electrophoresis of DNA fragments along with the proper molecular-weight standard on an agarose gel, suppose the outcome in a and b were observed. What might be the explanations for each outcome?

a. the gel is blank no bands no molecular weight standard

b. only the molecular weight standard is visible

a. something is wrong with the electrophoresis setup since the marker did not run. Perhaps the incorrect dye was used or was missed.

b. this must be a problem with the sample itself since the marker is present the electrophoresis worked. Either the sample wasn't loaded or the previous isolation step of the sample was not successful and the gel helps to visualize this

A 6% solution of 19:1 acrylamide:bis-acrylamide is mixed, de-aerated, and poured between glass plates for gel formation. After an hour, the solution is still liquid. What might be one explanation for the gel not polymerizing?

the nucleating agent or polymerization catalyst were not added to the gel solution

Why does DNA not resolve well in solution (without a gel matrix)?

Particle movement in solution is based on the charge/mass ratio. As the mass of DNA increases, slowing migration, the charge increases speeding migration, counteracting the effect of mass.

Name two dyes that are used to monitor migration of nucleic acid during migration.

Xylene cyanol and bromophenol blue ie tracking dyes