Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
  • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
  • G01N27/416—Systems
  • G01N27/447—Systems using electrophoresis
  • G01N27/44704—Details; Accessories
  • G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
  • G01N27/44734—Arrangements for investigating the separated zones, e.g. localising zones by thermal means

Definitions

• FIG. 1 shows the separation of an amplified DNA fragment (cystic fibrosis, CF, gene from a normal individual) in the absence (lower tracing) and presence (upper profile) of thermal denaturing gradients. In the lower tracing, separation occurs at constant temperature (45 * C) and in presence of chemical denaturants (6 M urea). The peaks eluted between 27 and 35 min represent oligonucleotide primers. The normal amplified DNA is eluted as a single peak (labelled W t /W t ) between 58 and 60 min.
• Fig. 3 shows the analysis of a set of intermediate melting fragments, amplified from CF patients heterozygous for different mutations in exon 11 of the CFTR gene: 1717-1G —> A (panel A); G542X (G —> T at 1756; panel C) and 1784delG (panel D) with their respective normal control (panel E). All mutants exhibit the characteristic four-peak profile, vs. a single band in the control. As shown in the temperature profile of panel E, these mutants are intermediate melters, with T ⁇ 's in the 56.5 to 57.8*C range.
• Fig. 4 shows the optimized condition set up for a higher melting fragment, amplified from a CF patient homozygous for the M1V mutation (A --> G transversion at position 133 in exon 1 of the CFTR gene).
• the panel shows the electropherogram of the sample injected at a constant temperature plateau (65'C), constant denaturant buffer, but in the absence of a temperature gradient.
• 65'C constant temperature plateau
• the group of peaks eluting from 35 to 48 min corresponds to unpurified primers with an without GC-cla ps.
• the insert shows the optimized separation in a 65 to 67 * C gradient with a slope of O.l'C/min: the correct spectrum of four bands is now obtained.
• the temperature is the one truly existing inside the capillary and is precisely determined with the aid of computer programs developed by us (M.S. Bello, E.I. Levine & P.O. Righetti: Computer assisted determination of the inner temperature and peak correction for capillary zone electrophoresis. J. Chromatogr. 652, 1993, 329-336). Use of masticated and "chain-transfer" polyacrylamides.
• Fig. 5 shows the progressive decrements of viscosity and average molecular mass of polyacrylamides during the mastication process by sonication.
• the viscosity has been measured with a Bohlin VOR rheo eter (Bohlin Rheology, Lund, Sweden), whereas M has been determined by gel permeation (HPLC Waters' 590 Solvent Delivery System, equipped with two Waters Ultrahydrogel columns and with a differential refractometric detector R401 against polyethylene glycol standards.
• Such reduced viscosity allows the injection into the capillary of much more concentrated solutions of polyacrylamides (up to 10%), which in turn permit optimization of resolution in the DNA size interval (typically from 100 to 500 bp) most interesting for the screening of genetic mutations via analysis of amplified DNA fragments.
• Even better separations can be achieved by polymerization in presence of chain-transfer agents (e.g., 3% isopropanol) coupled to elevated temperatures, a process which generates chains of further reduced lengths and viscosities.
• Fig. 5 shows viscosity measurements as a function of polymer concentration obtained by polymerization in presence of "chain transfer" agents at 35 * C and at 70 * C.
• the viscosity has been measured with a Bohlin VOR rheometer (Bohlin Rheology, Lund, Swden).
• Bohlin Rheology Lund, Swden
• the drastic viscosity reduction at high temperatures is due to formation of short chains (M r of only 180000 Da at 70 * C, as opposed to M r cf 450000 Da when polymerizing at 35'C).
• the viscosities of polyacrylamides polymerized at 35 or at 70 * C are markedly different.
• a strong decrement of viscosity is obtained (e.g., in an 8% polymer solution, the viscosity diminishes from 450 mPa.s to barely 120 mPa.s).
• This strong viscosity decrement is due to a marked reduction in average chain length, which diminishes from 430000 Da (when polymerizing at 35 * C) to only 180000 Da at 70 * C.

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• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Molecular Biology (AREA)
• Chemical & Material Sciences (AREA)
• Biochemistry (AREA)
• Electrochemistry (AREA)
• Physics & Mathematics (AREA)
• Analytical Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Health & Medical Sciences (AREA)
• General Physics & Mathematics (AREA)
• Immunology (AREA)
• Pathology (AREA)
• Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
• Saccharide Compounds (AREA)
• Investigating Or Analysing Biological Materials (AREA)
• Addition Polymer Or Copolymer, Post-Treatments, Or Chemical Modifications (AREA)

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• 1994
  • 1994-09-12 IT ITMI941864A patent/IT1271005B/it active IP Right Grant
• 1995
  • 1995-09-11 WO PCT/EP1995/003561 patent/WO1996008715A1/en not_active Application Discontinuation
  • 1995-09-11 JP JP8509892A patent/JP3015106B2/ja not_active Expired - Lifetime
  • 1995-09-11 EP EP95932707A patent/EP0781411A1/de not_active Withdrawn
  • 1995-09-11 AU AU35652/95A patent/AU689648B2/en not_active Ceased

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