Capillary Electrophoresis

Why are PCR products prepared for capillary electrophoresis by rapid heating and cooling?

• Ensure that the 2 complementary DNA strands are separated and denatured

Why is formamide used to dilute PCR products prior to electrophoresis and why should it have a low conductivity?

• to help denature the double stranded DNA, reduce the salt levels and aid in the electrokinetic injection process

• Low conductivity to minimize competition for injection and improve resolution

  • helps maintain single-base-pair resolution

What is the chemical composition of POP?

Linear uncross-linked dimethyl polyacrylamide with a high concentration of urea

Which POP is used in the lab?

POP-4 for STR typing

How is POP-4 different than POP-6 ?

POP-4 has a lower concentration of polydimethylacrylamide, 4% and is used for STR typing in our lab and POP-6 has 6% and is used for higher resolution to meet single-base resolution

What is the role of buffer in an electrophoresis system?

• Dissolve polymer to stabilize and solubilize the DNA and provide charge carriers for the electrophoretic current

What is the composition of the Applied Biosystems Genetic Analyzer buffer?

100 mmol/L N-tris-(hydroxymethyl)-methyl-3-aminopropane-sulfonic-acid (TAPS) and 1 mmol-L EDTA

What is the running temperature used at our laboratory for capillary electrophoresis? Why is it important to maintain a consistent running temperature?

• 60C, to reduce secondary structure in DNA

• Important as temperature variations can have profound effects on allele migration

  • Can cause secondary structures that alter migration of DNA fragments

How are negatively charged DNA molecules introduced onto the capillary

• Electrokinetic injection

  • Formamide solution added to DNA samples to denature double stranded DNA into single strands

  • Size marker added to allow accurate sizing of DNA fragments during analysis

  • Samples denatured using heat and snap cooled on ice to maintain single stranded separation

One end of capillary is placed inside well containing DNA samples and other is submerged in buffer reservoir. High voltage is applied across capillary, creating an electric field that causes (-) DNA to migrate from sample well into capillary toward (+) electrode. Injection is complete, capillary end moves back into buffer reservoir and higher separation voltage is applied which drives DNA fragments through capillary.

Describe how differently sized PCR products are separated as they migrate through the polymer in the capillary

• Based on their ability to move through the polymer matrix under the influence of an electric field

• DNA molecules (-) migrate from (-) cathode to (+) anode

What is the benefit of having a dynamic coating on the inner wall of a capillary?

Dynamic coating on the inner wall of a capillary helps prevent EOF in DNA separations. EOF is electrosmotic flow. Dynamic coating prevents EOF by coating the inner capillary wall to prevent exposure of (-) charges along the way build of ions (+)

What is the functional importance of the internal sizing standard?

• Reference for determining the precise size of unknown DNA fragments in the sample

• Consisted of DNA fragments on known lengths

• Helps convert time (s) into base pair

FGL uses what size calling method

Southern method