FR AB - Science Reference

ABRF 2001 ABSTRACTS
P9-T
A comparison of alternative methods for sequencing through
difficult templates.
D.A. Bintzler, Y. Song; Univ. of Cincinnati, 231 Bethesda Avenue,
Cincinnati, OH 45267-0524
Template DNA that has a seventy percent or greater content of guanosine
and cytosine bases (GC rich) has been a potential problem for automated fluorescent
DNA sequencing. Two well-known strategies for improving the base
read length of the GC rich template include the addition of dimethyl sulfoxide
(DMSO) or betaine to the PCR amplification of the template. Applied
Biosystems Incorporated (ABI) has offered a third method with dGTP big dye
chemistry. The dGTP chemistry has been significantly effective for extending
through templates that contain a chain of ten or more guanosine bases.
The dRhodamine chemistry (ABI) has also been effective for some problematic
templates. However, no single method has been effective for all problematic
templates in this facility. Therefore, research has continued to find an
alternative method. A PCR additive available from Qiagen, Q buffer, was
introduced as a potential method for improving the base read length of the
GC rich template. Preliminary studies showed that Q buffer had improved
PCR extension through difficult templates that were not successfully
sequenced with the other methods previously mentioned. However, a thorough
investigation of the Q buffer compared to DMSO, betaine, dRhodamine
and dGTP big dye chemistry was required. The results of the investigation
are reported here.
P11-M
Integration of new sequencing technologies into the W.M. Keck
Biotechnology/HHMI Biopolymer Lab. at Yale.
K.M. Hager; Yale Univ., 295 Congress Ave., New Haven, CT 06536
In the past year, we have successfully integrated the Applied Biosystems 3700
DNA Sequencer into our DNA sequencing service. This machine utilizes 96
capillaries and a proprietary polymer rather than polyacrylamide slab gels to
separate fluorescently-labeled DNA termination fragments. Because of 3700’s
unattended robotic sample loading from as many as four sample plates per
operator interaction, we have increased the lab’s overall sequencing throughput
by 25% with no increase in staff and lowered the average sample turnaround
time. In addition, the 3700’s automation has allowed us to initiate
Genescan and SNAPSHOT single-nucleotide polymorphism (SNP) detection
assays as new services. At present, about 75% of our total DNA sequencing
samples are run on the 3700 with the remainder run on one 377 (slab gel).
About 2–5% of the samples run on the 3700 give a poor separation and these
samples are rerun on the 377. Such poor separations are due to either
improper loading of the separation polymer into the capillary or overloading
of the capillary with excessive amounts of DNA. On the vast majority of
runs, our pgem standards yield 650–750 bases of sequence data at �99%
accuracy (POP-6 polymer).
Recently, we have begun an evaluation of Pyrosequencing, a non-electrophoretic
DNA sequencing technology using an enzyme coupled assay
where light is ultimately generated by luciferase from PPI released following
nucleotide incorporation (Science 281, pp. 363–364 (1998)). Our initial applications
for Pyrosequencing will include sequence tag (20–40 base) determination
and SNP detection.
POSTER ABSTRACTS
190 JOURNAL OF BIOMOLECULAR TECHNIQUES, VOLUME 11, ISSUE 4, DECEMBER 2000
P10-S
Fragment and sequence analysis in Portugal: a survey of facilities
and applications.
L.M. Souto; Univ. de Aveiro, Portugal, Departamento de Biologia,
Universidade de Aveiro, Aveiro, Aveiro 3810 Portugal
Since 1992, with the first unit of Automated Sequencers starting to work in
Portugal, a significant improvement in the number of equipments and institutions
dealing with sequencing and fragment analisys has been registered.
The leading groups are the forensic institutes which deal with a heavy routine
work, namely in paternity cases. These groups are using very standardised
techniques and markers, in accordance to the EDNAP (European DNA
Profiling Group) and the Portuguese and Spanish Working Group of the ISFH
(GEPISFH).
Recently, the universities as well as some private research and diagnostic labs
began to use DNA Sequencers, opening the spectrum of applications.
We present in this poster the state of the art of Automated Sequence and
Fragment Analysis in Portugal and its relationship with other iberian and
latin-american countries, with a special emphasis onto the forensic genetics
field.
P12-T
Investigation of the entire spectrum of genes induced by staphylococcal
enterotoxin B in human lymphoid cells using differential
display-PCR.
C.A. Mendis, C. Sanchez, R. Das, M. Jett; Walter Reed Army Inst. of Res.,
503 Forney Drive, Silver Spring, MD 20910
Since its introduction in 1991, Differential Display has evolved into a powerful
and efficient technique in analyzing differentially expressed genes in
various cells under altered conditions. Here we used differential display not
only to screen the entire gene population in human lymphoid cells induced
by SEB but also to understand the specificity of the expression pattern of
altered genes by comparing it to the expression by Cholera Toxin (CT). In
order to approach our goal in a systematic manner as well as to increase the
efficiency of cDNA product recovery we used a set of arbitrary and anchored
primers to further subdivide the gene population. Out of the 750 altered
genes, at least 200 genes showed a SEB specific expression pattern when
compared to CT exposure. The altered genes were then subjected to a high
throughput cloning procedure and sequenced. Out of the 250 genes that
were sequenced, 150 genes matched with available sequences in the Gene
Bank and EMBL databases. Unique to SEB are genes involved in ancillary
functions as adhesion molecules, regulators of vascular tone, wound healing,
inflammation, heat shock, cell death and T-cell proliferation. Altered genes
that matched with known sequences were verified by northern blots, RT-PCR
or real time PCR. Genes that were specific for SEB were then placed on glass
chips for use with other cell lines or to understand the gene expression profile
of other toxins. In the quest for finding a set of genes that can be used
as surrogate markers, differential display has shown to be a proven technique
as it has the capability to look at the whole gene spectrum irrespective of
wide use or availability of individual genes.