Principles Of Ttge

Temporal temperature gradient gel electrophoresis (TTGE) was first introduced by Yoshino et al.[13] in 1991. It is based on the difference in the sequence-specific melting behavior of the normal and mutant DNA in a temporal temperature gradient that gradually increases in a linear fashion over the duration of the electrophoresis (Fig. 1). In TTGE, the denaturing environment is formed by a constant concentration of urea in the polyacrylamide gel and a temporal temperature gradient (0.5-3°C/hr). During the course of electrophoresis, the gradual temperature increase causes the double-stranded DNA to become partially denatured, and the electrophoretic mobility of the partially denatured DNA decreases. As a result of the different melting behavior, mutant and wild-type molecules are separated on the gel as they begin to denature at different temperatures (Fig. 1). A homozygous mutation shows band shift and a heterozygous mutation shows multiple bands because of the presence of two homo-duplexes and two heteroduplexes at optimal separation conditions. Usually, an AT base pair to GC base pair change results in band shift-down and the GC pair to AT pair change results in band shift-up. This is because three hydrogen bonds in GC pairs hold the double-stranded structure tighter than the two hydrogen bonds in AT pairs. TTGE differs from TGGE in that TGGE has a fixed

} Heteroduplex } Homoduplex

Fig. 1 Principles of TTGE. The PCR products are denatured and reannealed gradually to form homoduplexes, and heteroduplexes if there are mutations. During electrophoresis, the duplexes become partially denatured due to the temperature gradient. Based on the melting behavior of the duplexes, the partially denatured duplexes become separated while the temperature rises. (View this art in color at www.dekker.com.)

30 sec and slowly cooled to 45°C for a period of 45 min at a rate of 1°C/min. The samples containing reannealed homoduplexes and heteroduplexes are maintained at 4°C until loading onto the gel. TTGE is performed on a BioRad D-Code apparatus. Two back-to-back 20 cm x 20 cm x 1 mm 4.5-6% polyacrylamide (acrylamide/ Bis = 37.5:1) gels are prepared in 1.25 x TAE buffer containing 6 mol/L urea. The percentage of the gel depends on the size of the PCR product. Five microliters of denatured and reannealed PCR products are mixed with equal volume of 2 x loading buffer (70% glycerol, 0.05% bromophenol blue, and 0.05% xylene cyanol) and loaded onto the gel. The electrophoresis is carried out at 110145 V for 4-7 hr at a constant temperature increment of 1-2°C/hr. The temperature range for the electrophoresis depends on the melt curve of the DNA fragment determined by computer simulation using MacMelt (or WinMelt) software (Bio-Rad Laboratories). In addition, the melting temperature decreases by approximately 2°C for each molar of urea used. The gels are stained in 2 mg/L ethidium bromide for 5 min and imaged with a digital charge-coupled device (CCD) gel documentation system. DNA fragments showing alterations in banding patterns are sequenced to determine the nucleotide change. To confirm if a mtDNA mutation is homoplas-mic or heteroplasmic, usually a second method, such as PCR/allele-specific oligonucleotide (ASO), dot blot, or PCR/restriction fragment length polymorphism (RFLP) analysis is used.[15,16]

} Heteroduplex } Homoduplex

Fig. 1 Principles of TTGE. The PCR products are denatured and reannealed gradually to form homoduplexes, and heteroduplexes if there are mutations. During electrophoresis, the duplexes become partially denatured due to the temperature gradient. Based on the melting behavior of the duplexes, the partially denatured duplexes become separated while the temperature rises. (View this art in color at www.dekker.com.)

temperature gradient from top to bottom of the gel.[14] In TTGE, the temperature at any location of the entire gel is the same at any given time, but changes with respect to time (temporal temperature). Thus it is easier to modulate the temperature during electrophoresis and provide a broader separation range that leads to a much higher sensitivity of detection. Because the denaturant in TTGE is the temperature, there is no difficulty in the preparation of a chemical denaturant gradient gel. The size of the DNA fragments to be analyzed on TTGE can be as large as 1 kb. A GC clamped primer is not a requirement.

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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