UNIT 6. NUCLEIC ACID ELECTROPHORESIS

electrophoresis

is the movement of molecules by an electric current.

Nucleic acids

have a negative charge so they move towards the positive pole.

tubes, slab gels, or capillaries.

Electrophoresis can be performed in

1. agarose gel

2. polyacrylamide gel

2 NA gel systems

agarose

Gel systems:

• uses a _______ a polysaccharide from seaweed

smaller pores

Gel Systems:

• Larger concentrations of agarose produce _________ in the gel making it harder for nucleic acids to move.

Pulse Field Gel Electrophoresis

• Used for resolving very large fragments of DNA

• DNA goes forward and backward, alternating, circular, and polygon, allowing better resolution of large fragments.

Pulses of current

Pulse Field Gel Electrophoresis:

• _________ from alternating sides of the gel are used.

Polyacrylamide Gel

Gel systems:

• Best for smaller DNA fragments.

• Used for sequencing nucleic acids, mutation analyses, nuclease protection assays, and other applications requiring the resolution of nucleic acids down to the single-base level.

Acrylamide;

cross-linker methylene bisacrylamide;

polyacrylamide

____________, in combination with the _____________, polymerizes into a gel that has consistent resolution characteristics, the ____________

proteins

Polyacrylamide was originally for _____

T

T or F
Preparation of PAGE gels is more complicated than AGE gels

Capillary electrophoresis

Gel system:

• A polymer is placed inside inside capillary tubes and the sample moves through there.

• Sample has to be denatured.

• Less sample, immediate visualization, increased sensitivity, less work, less time, but much more costly.

buffer;

electric current;

buffering capacity

In order for nucleic acids to migrate properly, the gel system must be immersed in a ______ that conducts the ________ efficiently in relation to the __________

1. Tris borate EDTA (TBE)

2. Tris phosphate EDTA (TPE)

3. Tris acetate EDTA (TAE)

3 Tris buffers used in electrophoresis

0.089 M Trisbase

0.089 M boric acid

0.002 M EDTA

TBE composition

0.089 M Trisbase

1.3% phosphoric acid

0.002 M EDTA

TPE composition

0.04 M Trisbase

0.005 M sodium acetate

0.002 M EDTA

TAE composition

TAE

most commonly used Trisbase

Buffer additives

__________ are often used to modify sample molecules in ways that affect their migration.

Gel loading buffer (GLB)

Samples must be first mixed with ________________

tracking dye & density agent

GLB contains (2)

Density Agent (Ficoll, sucrose, glycerol)

increases the density of the solution as compared with the electrophoresis buffer

tracking dyes (bromophenol blue or xylene cyanol green)

• are used to monitor the progress of the electrophoresis run.

• migrate at specific speeds in and usually run ahead of the smallest fragments.

F

T or F

Tracking dyes affect the separation of the sample DNA.

F

they only migrate along the DNA but do not bind to it

T or F

Tracking dyes help locate where the DNA is in the gel by binding onto the DNA.

Nucleic-Acid specific dyes

Used to visualize DNA/RNA

1. Intercalating agents: Ethidium bromide (EtBr)

2. SYBR green dyes

3. Silver stains

3 Nucleic-Acid specific dyes

Intercalating agents

3 Nucleic-Acid specific dyes

• intercalate, or stack, between the nitrogen bases in double-stranded nucleic acid.

Ethidium bromide (EtBr)

3 Nucleic-Acid specific dyes

• When exposed to UV light, it fluoresces (orange glow).

• The more DNA present → the brighter the band.

• neutrogen, carcinogen and high contamination

SYBR Green

3 Nucleic-Acid specific dyes

• Binds to the minor groove or sides of DNA

• Fluoresces under blue or UV light.

• relatively safe, non-toxic, non-contaminating

Silver stain

3 Nucleic-Acid specific dyes

• Staining solution interacts with nucleophilic groups in DNA/RNA.

• Silver ions (Ag+) gets reduced and deposits directly where nucleic acids are located.

• Produces dark brown/black bands.

• Extremely sensitive (detects very small amounts of DNA).

• Does not require UV light.

AGE of Genomic DNA

• heavy, long smears with intense bright bands

ReliaPrep™ Large Volume

Maxwell® 16 LEV

Maxwell® HT 96 gDNA

Wizard® Genomic

QIAamp® DNA Blood

RNA contaminationD

• smaller fragments deposited at the bottom of the gel

• blurred smears at the bottom

DNA degradation

• smear only with no bright bands of DNA

• shearing

AGE of Plasmids

• circular

• linear

• supercoiled

• nicked

Plasmid conformations

supercoiled plasmid

AGE of Plasmids

• always the band at the bottom (not RNA contamination)

Genomic DNA contamination

Open Circular / Covalently Bounded

Linear Plasmid

Supercoiled Plasmid

RNA Contamination

AGE of RNA

• 2 very distinct bright bands at 18S and 28S (for eukaryotes) or 16S for (prokaryotes)

2 ribosomal subunits (1 large, 1 small subunits)

AGE of RNA:

• The 2 very distinct bright bands at 18S and 28S are representative of

Molecular weight markers

Consists of nucleic acid fragments of known sizes used to identify the sizes of unknown nucleic acid bands

• 1Kb plus DNA ladder

• 100 bp DNA ladder

• Lambda-HindIII DNA ladder

• RNA marker

different types of markers

• Main application is resolve nucleic acid molecules.

• DNA sequencing

• Routine check for PCR products

• Routine check for cloning

• Routine check for enzyme digestion

• Paternity testing

Application of nucleic acid gel electrophoresis