resolution and detection of nucleic acids (part 3 of exam 1)

resolution

the process of reducing or separating something into its components

electrophoresis

movement of molecules through support media by electrical current

under an electrical current, DNA and RNA will migrate towards what pole?

positive pole (anode)

what is needed to run an electrophoresis

sample

support media (agarose or polyacrylamide gels)

buffer w/ additives

electrophoresis chamber

power source

concentration of agarose gel dictates what in the gel?

size of the pores

what is the range of pore size in agarose gel?

100 to 300 nm

small pieces of DNA (50 - 500 bp) are resolved on what kind of gels?

more concentrated: 2-3%

larger fragments of DNA (2,000 - 50,000 bp) are resolved on what kind of gels?

lower concentrations: 0.5-1%

what is the limit for agarose gel size?

<0.5% and >5%

relationship of agarose gel size to nucleic acid size

the more concentrated the gel, more resistance, less movement

when is pulse field gel electrophoresis (PFGE) used?

for very large pieces of DNA (50,000 to >250,000 bp)

pulse field gel electrophoresis (PFGE)

pulses of current applied to gel in alternating dimensions

“duck and weave”

applications of pulse field gel electrophoresis (PFGE)

resolution of chromosomes

bacterial typing

epidemiological studies

whole DNA analyses

components of pulse field gel electrophoresis (PFGE)

switch interval

• PFGE applies current in alternating directions at specific times

sample runs can take >24 hrs

cooling unit required

what are polyacrylamide gels (PAGE) made of?

polymerized acrylamide + methylene bisacrylamide

what is needed to polymerize polyacrylamide?

catalyst: APS and TEMED

when are PAGE gels used?

small DNA fragments, ssDNA, RNA, proteins

capillary electrophoresis

sample in thin glass capillary, two buffer solutions and anode

faster runs and lower cost

costly instrumentation

buffer

solution of weak acid and its conjugate base

purpose of buffers during electrophoresis

carry the current and protect samples

what happens in electrophoresis when there is a high buffer concentration?

increases conductivity (more current/flow) of electrophoresis system, generating more heat

• lessen gel stability

what buffers are preferred for electrophoresis?

Tris, TBE, TAE

buffer additives

modify molecules in sample so it affects their migration

denaturing agents: formamide and urea break H bonds hindering complementary sequences from reannealing

in what direction are agarose gels run?

horizontally

in what direction are polyacrylamide gels run?

vertically

what is the voltage for running gel and polyacrylamide electrophoresis?

80-150 for 1-2 hrs

sample preparation

~10 ng of nucleic acid can be visualized

tracking dyes like bromphenol blue

density agent: Ficoll, sucrose, glycerol

DNA control ladder (molecular standard)

when is electrophoresis terminated?

when tracking dye approaches end of gel

what does a density agent like Ficoll, sucrose, or glycerol do for sample preparation?

increases density of sample and allow it to sink into wells when dispensed

detection systems

fluorescent dyes and silver stain

fluorescent dyes

intercalating agents: EtBr
minor groove-binding dyes: SYBR green

after electrophoresis, is sample ruined?

can purify nucleic acids from agarose gel by cutting out band of interest