MODX MIDTERM RESOLUTION AND DETECTION OF NUCLEIC ACIDS

• Electrophoresis


  is the movement of molecules by an

  electric current
• electric current


  Under an __, DNA and RNA will migrate toward the positive pole (anode
• Gel Matrix


  used to limit migration and contain the migrating material
• Arne Tiselius


  developed an electrophoresis appaatus while studying resolution diffusion and adsorption of proteins in naturally occurring porous silicate particles
• Oliver Smithies


  later found that if he heated about 15 grams of starch in 100 mL of his electrophoretic medium and allowed it to cool, he could make a gel matrix with resolving properties similar to a paper electrophoresis method he had previously devised
• Smithies and Poulik


  described the resolution of 20 serum protein comonents using a two-dimensional electrophoresis system with paper in one dimension and starch in the other
• Pulsed-Field Gel Electrophoresis


  For these very large DNA molecules, pulses of current applied to the gel in alternating dimensions enhance migration
• field-inversion-Gel-Electrophoresis


  simplest approach to this method (PEGE) is
• PFGE-instruments, switch-interval


  __ will then apply current in alternating directions at specifi c times (called the __ ) that are set by the operator
• polyacrylamide-gel-electrophoresis-PAGE)


  Very small DNA fragments, single-stranded DNA, RNA,

  and proteins are best resolved on polyacrylamide gels in __
• Polyacrylamide Gels


  allows precise control of the polymer properties and higher resolution than can be achieved with agarose
• polyacrylamide gels


  With single-base resolution, __ are used for nucleic acid sequencing, mutation analyses, nuclease protection assays, and other applications requiring high resolution of nucleic acids
• Preformed gels


  __ are the most convenient because the procedure for preparation of acrylamide gels is more diffi cult than that for agarose gels
• agents ammonium persulfate (APS) plus N,N,N ′ ,N ′ -tetramethylethylenediamine (TEMED)


  catalysts of polyacrylamide gels
• CAPILLARY ELECTROPHORESIS


  separation of organic chemicals such as pharmaceuticals and carbohydrates
• capillary electrophoresis


  For these applications, __ has the advantages of faster analytical runs and lower cost per run than other separation methods, also used for the separation and anal sis of nucleic acids
• Fused silica


  Fused silica is used as the capillary tube because it is the most transparent material allowing for the passage of fluorescent light
• polyimide coating


  The fused silica is covered with a __ for protection, except for an uncoated window where laser light excites the fl uorescent mol cules attached to the fragments as they pass a detec ion device
• Fluorescent labels


  are covalently attached to nucleic acids to be separated by capillary electrophoresis
• formamide


  denatured nucleic acid in buffer containing __ are introduced to the capillary, which is held at a dena uring temperature, usually 50°C to 60°C
• buffer system


  The purpose of a __ is to carry the current

  and protect the samples during electrophoresis
• Henderson-Hasselbalch equation


  The amount the pH of a buffer will differ from the pKa is expressed as the
• Buffer additives


  modify sample molecules in ways that

  affect their migration
• pH drift


  decrease of pH at the cathode [-] and an increase of the pH at the anode [+])
• horizontal or vertical


  Gel electrophoresis is performed in one of two confor

  ations, __ or __
• submarine gels


  hori ontal gels are sometimes referred to as__
• tracking dye


  Prior to loading the sample containing isolated nucleic

  acid onto a gel, __ and a density agent are added to the sample
• density agent


  the __ (Ficoll, sucrose, or glycerol) increases the density of the sample as com

  ared with the electrophoresis buffer
• bright-blue/ royal-blue

  color of xylene cyanol
• Ethidium bromide (3,8-diamino-5-ethyl-6-phenylphenan hridinium bromide [EtBr


  _ is one of these agents and was the most widely used dye in early DNA and RNA analyses
• DNA, DNA or RNA


  SYBR green I used for __ staining; SYBR green II, for single-stranded __ stain ng; and SYBR gold
• SYBR green


  __ is the preferred dye for real-time PCR methods
• silver stain


  Another sensitive staining system originally developed

  for protein visualization is __
• silver diamine, silver nitrate


  Of the two procedures, __ is best for thick gels, whereas _ is considered to be more stable
• TBE, TPE, TAE

  most commonly used for buffer electrophoresis