To investigate the fragment length-dependent sequencing bias, we prepared a restriction digest
of [lambda] DNA (Invitrogen).
Our PCR, restriction digest
, and electrophoresis protocols were adapted from Gruntzig et al.
Four distinct phylogenetic lineages of human serotype III GBS have been identified by PFGE of restriction digest
patterns and designated restriction digest
patterns type III-1, III-2, III-3, and III-4 (11).
To quantify the proportion of the DNA that could be digested, we divided the density of the full-length PCR product after the restriction digest
by the density before the restriction digest
The Agilent 2100 bioanalyzer is now offered with five LabChip kits including three for the high-resolution analysis of DNA samples, such as PCR products and restriction digest
fragments, one for the analysis of RNA samples typically used in gene expression applications, and the new protein assay.
pseudomallei were typed by PFGE of an XbaI restriction digest
of bacterial chromosomal DNA (Figure).
An aliquot of the restriction digest
is taken for PFGE analysis as a control for DNA preparation; the remaining prepared DNA molecules are analyzed with the DLA system.
These instruments are being currently used for a wide variety of applications including microsatellite analysis, restriction digest
sample quality checks as well as oligonucleotide quality checks.
Each sample was run in triplicate using one lane for the restriction digest
and an adjacent control lane for the uncut DNA.
products were size fractionated by electrophoresis on a 2% agarose gel with 1 mg/L ethidium bromide for 20 min at 200 V and detected directly under ultraviolet light.
One microliter of undiluted restriction digest
was amplified in the second PCR in a 20-[micro]L volume in 1 x PCR buffer containing 1.
Evaluation of 100 human DNA samples for factor V Leiden by both the restriction digest
and the fluorescence protocols yielded identical genotyping results by both methods: 60 samples were wild-type, 37 were heterozygous, and 3 were homozygous for the factor V Leiden mutation.