Instruction manual

Fig. 2.1. An example of DNA melting properties in a denaturing gradient gel. At a low concentra-

tion of denaturants the DNA fragment remains double stranded, but as the concentration of denatu-

rants increases the DNA fragment begins to melt. Then, at very high concentrations of denaturants, the

DNA fragment can completely melt, creating two single strands.

The thermodynamics of the transition of double stranded DNA to single stranded DNA

have been described by a computer program developed by Lerman,

232

based on the statistical

mechanical principles and algorithms developed by Poland

233

and the nearest-neighbor

base-pair doublet parameters introduced by Gotoh and Tagashira.

234

There are programs

available which calculate the theoretical DNA melting profile of a given sequence. Bio-Rad

offers Macintosh

computer program, MacMelt

™

software, which calculates and graphs

theoretical DNA melting profiles.

Applications of the D GENE system are varied, but its primary purposes are screening for

mutations after in vitro or in vivo mutagenesis, screening for tumor material or acquired

mutations, analysis of candidate genes for possible mutation, analysis of inherited disease,

analysis of new genes, analysis of polymorphism, and marker screening. Other applications

include regulatory protein-nucleic acid complex formation, confirmation of the accuracy of

PCR amplified reaction mixtures, and assay of PCR induced mutations in cloned sequences

without sequencing.

Single strands

Double strand

Partially melted

“wild type”

Partially melted

“mutant”

Wild Type

Mutant

100%

Denaturant

Electrophoresis