The gels resulting from PCR amplification of conserved bacteria domain sequences followed by TTGE (evaluation of total bacterial diversity) were examined for bands at 100 possible positions.
In the bacterial TTGE, it is not possible to discriminate between individual species of bifidobacteria.
A variety of methods for mtDNA mutation screening have been published, such as DHPLC (17) and TTGE (20).
Enhanced detection of deleterious mutations by TTGE analysis of mother and child's DNA side by side.
We have developed a TTGE method to detect mutations and polymorphisms in the [beta]-globin gene based on the melting profiles described previously by Ghanem et al.
For TTGE, the [beta]-globin gene was amplified as seven fragments (A, B, C, D, E, F, and G) using the primers described for DGGE by Ghanem et al.
1A), in theory TTGE should allow quantification of the proportion of both heteroplasmic species, in addition to its usual role in mutation detection.
To investigate the possibility of using TTGE as a quantitative method, we constructed "artificial heteroplasmies" by mixing DNA samples obtained from blood from two individuals known to differ in sequence (among the amplified segment) by a single nucleotide, T3197C, in the [tRNA.sup.leu(UUR)] gene, or by a 9-bp insertion in the small noncoding area between COX2 and [tRNA.sup.lys] (corresponding to triplication of the sequence from nt 8272 to nt 8280, whereas duplication of the 9-bp sequence is the norm) (4).
The primers used in PCR amplification, the sizes of PCR products, temperature ranges, and ramp rates of the TTGE analysis are listed in Table 1.
DISTINCTION OF HOMOPLASMIC AND HETEROPLASMIC MUTATIONS BY TTGE
is a heteroduplex detection method similar to denaturing gradient gel electrophoresis (DGGE).
has been shown to be a powerful tool for the detection of novel nuclear DNA mutations (8-10).