Genet 390 Topic 3

What is the basic principle of gel electrophoresis?

separates molecules based on size

to run a gel you need...

- a sample (DNA/RNA/protein)

- a gel (polyacrylamide or agarose)

- a buffer

- electric field

- a visualization molecule

benefits of agarose gels

- cheap

- quick

- easy

- non toxic

- easy to melt and repolymerize (microwave)

what is agarose made of?

- a linear polymer of repeating agarobiose (a disaccharide)

- polymerizes spontaneously

- acts as a sieve to slow down larger molecules

the velocity of nucleic acids through an agarose matrix depends on...

- charge

- mass

- shape

effect of agarose gel %

higher % gels resolve smaller bands better. Lower % gels resolve larger bands better.

0.4% agarose gel range of separation

2.5-30kb

0.8% agarose gel range of separation

1-15kb

1% agarose gel range of separation

0.5-10kb

- starting point for unknown sequences

- good for separating medium sized DNA molecules

2% agarose gel range of separation?

0.1-2.5kb

what needs to be done to a dsDNA sample in order to run it on a gel?

- don't have to do much (typically already linear)

- already has a constant negative charge per unit length (on 5'PO4)

- constant mass per unit length

- plasmid and genomic DNA can be supercoiled so they run faster than the bp would suggest

buffer for DNA agarose gel electrophoresis

- TBE (Tris-Borate EDTA)

- TAE (Tris-acetate EDTA)

- Higher pH bc DNA is more stable in basic environments

how does an electrolytic cell work?

- "run to red"

- negative at top run towards positive at bottom

setting your voltage depends on...

- DNA size (2.5-10 volts/cm)

- type of buffer (TAE vs TBE)

- length of gel

- gotta be mindful of voltage and gel % so it doesn't melt

how does % and voltage relate for gels?

- lower % gel needs lower voltage

- smears can be indicative of too high a voltage (ran gel "too hot")

How do you "see" DNA?

1) Ethidium Bromide (EtBr)

2) SYBR Safe

how does EtBr work?

- fluorescent dye that intercalates into the DNA and also absorbs UV light

- abosrbs light best at 254nm but that damages DNA so 300nm is used

- intercalates every other bp (~50% of the DNA fluoresces)

- alters nucleic acid migration bc it has (+) charge and slows migration (but not by a ton)

- reemits orange light ~590nm

problems with EtBr

- carcinogenic (intercalating agent)

- using UV visualizer can burn you

- extended exposure of EtBr to treated DNA to UV light can cause DNA cleavage

- smaller bands have less fluorescence (less room for dye to intercalate)

SYBR safe advantages

- less chemical toxicity

- fewer disposal issues

- better sensitivity than EtBr

- low background staining

- stains RNA and DNA

SYBR safe disadvantages

- expensive

- stain useful for only 1 gel

how to estimate length of DNA fragments run on a gel

- "eyeball method"

- or plot log10 length (bp) vs migration distance (mm)

- or plot length (bp) vs 1/m (distance migrated)

- compare length migrated to standard curve of the gel

how to estimate mass of DNA fragments run on a gel

- length is proportional to mass for DNA

- can compare EtBr brightness with linear DNA ladder fragments (dye companies provide mass of each fragment)

how does running an RNA gel differ from running a DNA gel?

- RNA has secondary structure that must be denatured before running a gel

- denature RNA using formamide and formaldehyde in loading buffer

- heat sample to ~65ºC for 15 minutes

- use MOPs buffer pH 7

- requires RNA markers

- can still use EtBr to visualize tho

why do we use MOPs buffer instead of Tris-based buffers for RNA gels?

1) RNA is unstable at pH 8.3 (typical Tris based buffer pH)

2) Formamide/formaldehyde for H-bonds with RNA at pH7 but not at pH 8.3

3) formamide and formaldehyde will interact with Tris as well, often via H-bonds with Tris preferentially to bonding with RNA

what are the 3 forms of uncut plasmid DNA?

- OC= open circular (nicked dsDNA causes relief of supercoiling, least mobile)

- CCC= covalently closed circular

- SC = supercoiled (runs fastest)

why do you want SC plasmids for plasmid based work?

- represents intact and unaltered plasmid

- other forms may include cuts/nicks and DNA replication does not work across a nick

- BUT... to confirm the right plasmid we can use linearized plasmid to determine size

why might you want to extract a specific band from a gel?

- to purify a desired fragment for cloning, labelling, sequencing

- "band prep"

band prep procedure outline

1) dissolution

2) separation

3) wash DNA

4) Elute

band prep dissolution

- dissolve the gel with a little heat and buffer

band prep separation

- bind DNA to silica (+ charge) gel membrane under high salt conditions and pH

band prep wash

- high salt wash and ethanol to remove guanidine and salt but maintain DNA on membrane

why do you want to remove guanidine in the wash step?

- it denatures proteins and thus would affect downstream applications

band prep elution

- wash DNA with low salt concentration buffer

- most often we use a kit

what type of gel is used in PAGE electrophoresis?

polyacrylamide gel

* acrylamide is a neurotoxin (wear gloves!!!!!)

when do we use PAGE electrophoresis?

- used to separate smaller molecules like ssDNA, RNA, proteins

how does acrylamide polymerize?

- polymerizes into linear chains and then is crosslinked with bis-acrylamide (linker)

- crosslinking of the linear polymer determines pore size

what determines the final pore size of a polyacrylamide gel?

- 2 terms dictate the final pore size and therefore the size of the protein that can be separated on the basis of size (mass) alone

- %T and %C

- increasing %T and %C decreases pore size

what is %T?

- total acrylamide concentration per volume

- if you increase %T you decrease the pore size to separate smaller molecules

- proteins = 7.5-20% T

- ssDNA or RNA = 10% T

- "T" think total

what is %C?

- relative bis concentration of the total acrylamide

- as you increase %C (bis), you increase crosslinking and therefore decrease pore size

- "C" think crosslinker

%T and %C of typical PAG gel formulation?

20% T, 5% C

purpose of running ssDNA on PAGE gel?

- DNA sequencing

- oligonucleotide purification, etc.

what must be done to ssDNA to run it on a PAGE gel?

- must be denatured to migrate based on size (length) alone

how do we denature ssDNA?

1) urea treatment

2) heat (often with formamide)

3) run gels heated to ~70ºC

what is the purpose of running RNA on PAGE gels?

- separation of short RNA fragments up to ~300nt (eg. small regulatory RNAs)

- note if the RNA is >300nt, use an agarose gel

what needs to be done to RNA to run it on PAGE gel?

- RNA needs to be denatured to migrate based on size (length) alone

how do we denature RNA?

1) heat denature ~65ºC for 15 mins

2) formaldehyde + formamide

3) denature while gel running (have formaldehyde in gel ~15%)

how do we find a specific fragment in a sample?

- we let nucleic acid hybridization do the work by using probes

what are probes?

- short oligomers complementary to the sequence of interest

- labeled with some kind of visualizer (radioactivity, fluorescence, DIG/biotin)

caveats to buying probes

- can be expensive

- order sizes are often too large for typical applications

DNA probe labels that are commonly used

DIG-dUTP and Biotin-11-dUTP

what are the 2 locations for the label?

- body labelling --> throughout / within sequence

- end-labelling --> at 3'end

ingredients for body labelling?

- denatured dsDNA template

- klenow DNA pol

- randome primers (hexanucleotide) or identified primers

- 3' unlabelled dNTPs and labelled r/dNTPs

what's Klenow?

- fragment of DNA polymerase that is frequently used in molecular biology

- also called the large fragment of DNA pol I

- does not contain the 5'-3' exonuclease domain of DNA pol I

- this is preferred because you don't want to remove and sequences ahead of the polymerized strand

What are the Klenow dependent labelling techniques using DNA synthesis?

1) 5' overhang labeling

2) 3' overhang end labeling

3) blunt end labelling

how does 5' overhang end labeling work?

- see pg. 14

how does 3' overhang end labeling work?

- see pg. 14

how does blunt end labelling work?

- see pg. 15

what are the Klenow independent techniques not requiring DNA synthesis?

1) TdT-Terminal deoxynucleotide Transferase

2) PNK

what is TdT?

- Terminal deoxynucleotide Transferase

- aka homopolymer tailing

- TdT= DNA template indpendent polymerase which adds deoxynuclotides to the 3'OH terminus of dsDNA and ssDNA

- NOTE: a random # of nucleotides are added to each end of the DNA

- uses 3' end labeling for; RACE, TUNEL, Cloning, EMSA

what is PNK?

- polynucleotide kinase

- isolated from T4 bacteriophage

- can label 5' ends of the polynucleotides with radioactive phosphate using a phosphate exchange reaction

what is the best RNA probe?

Riboprobe

how does Riboprobe work?

- produces a ssRNA probe that is labeled with either 32P-UTP or a non radioactive marker like biotin or fluorescence

Riboprobe procedure

see page 16

Southern blot/ Northern blot/ Microarray

see pages 17-19