molbio: resolution and detection of nucleic acids

electrophoresis

is the movement of molecules by an electric current

negative to positive poles

Nucleic acid moves from the

resolution of fragments

The concentration of gel/buffer will affect the

horizontal or vertical format

Slab gel electrophoresis can have either a

pulsed field gel electrophoresis (PFGE ) systems

Very large DNA molecules are separated by

Field-inversion gel electrophoresis (FIGE), Transverse alternative field electrophoresis (TAFE), Contour-clamped homogenous electric field (CHEF), Rotating gel electrophoresis (RGE)

type of PFGE

Field-inversion gel electrophoresis (FIGE)

alternating positive and negative poles

Transverse alternative field electrophoresis (TAFE)

transverse angle reorientation of poles on a vertical

Contour-clamped homogenous electric field (CHEF)

alternating polarity in an electrode array

Rotating gel electrophoresis (RGE)

rotating gel with fixed poles

POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE)

Acrylamide in combination with a cross linker, methylene bis-acrylamide

ammonium persulfate (APS) plus N,N,N’,N’-tetramethylethylenediamine (TEMED), or light activation

Polymerization catalysts

1-kikobases molecule (0.1% difference)

PAGE Resolves 1-base pair difference in a

CAPILLARY ELECTROPHORESIS

Separates solutes by charge/mass ratio

Capillary gel electrophoresis

is used to separate nucleic acids

Linear or cross-linked polyacrylamide

are used for sieving

size

Separation is base on

rapid and automated

CGE is more ____ than slab gels

ELECTROKINETIC INJECTION

• Nucleic acid fragments to be separated are drawn to the mouth of the capillary with a transient positive charge

• Once collected, fragments migrate through the capillary to the positive pole

ELECTROPHORESIS BUFFERS

Carry current and protect samples during electrophoresis

use most often for DNA

Tris-borate EDTA (TBE), Tris-acetate EDTA (TAE), Tris phosphate EDTA (TPE)

10-millimolar sodium phosphate or MOPS buffer

used for RNA

buffer additives

can be used to modify sample molecules

formamide, urea

example of buffer additives

agarose gel

Horizontal or submarine gels are usually (but not always)

polyacrylamide gels

Vertical gels are usually (but not always)

combs

are placed in the gel before polymerization to leave wells in the cast gel for sample loading

ear

Wells are separated by an “__” of gel

.5% to 5% agarose, 3.5% to 20% polyacrylamide

proper gel concentration for sample size range

VISUALIZATION OF SEPARATED BANDS

Detect bands by staining during or after electrophoresis

ethidium bromide

for double-stranded DNA

SYBR Green or SYBR Gold

for single or double stranded DNA

silver stain

more sensitive for single or double stranded DNA or for RNA and proteins