Abstract: The SNaPshot® Multiplex System is a
primer extension–based method developed for the analysis of
single nucleotide polymorphisms (SNPs) (Figure 1). Through its
multiplexing capability, up to 10 SNPs can be analyzed in a single
reaction, regardless of their positions on the chromosome or the
amount of separation from neighboring SNP loci. The ability to use
unlabeled, user-defined primers allows researchers to
incorporate SNPs of interest cost-effectively. The Multiplex Ready
Reaction Mix (included in the system) helps ensure robust,
reproducible analyses of multiplexed samples. Researchers can analyze
more than 23,000 SNP genotypes per day on just one 3730xl Genetic
Analyzer.
White paper que descreve o uso da técnica de Análise de Fragmentos para o mapeamento gênico
Abstract: BAC fingerprinting provides an efficient and cost-effective method of characterizing large genomic fragment libraries for genome sequencing, positional cloning, and physical mapping efforts.
High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis.Abstract: We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-editing computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10.
High-throughput fingerprinting of bacterial artificial chromosomes using the snapshot labeling kit and sizing of restriction fragments by capillary electrophoresis.Abstract: We have developed an automated, high-throughput fingerprinting technique for large genomic DNA fragments suitable for the construction of physical maps of large genomes. In the technique described here, BAC DNA is isolated in a 96-well plate format and simultaneously digested with four 6-bp-recognizing restriction endonucleases that generate 3' recessed ends and one 4-bp-recognizing restriction endonuclease that generates a blunt end. Each of the four recessed 3' ends is labeled with a different fluorescent dye, and restriction fragments are sized on a capillary DNA analyzer. The resulting fingerprints are edited with a fingerprint-editing computer program and contigs are assembled with the FPC computer program. The technique was evaluated by repeated fingerprinting of several BACs included as controls in plates during routine fingerprinting of a BAC library and by reconstruction of contigs of rice BAC clones with known positions on rice chromosome 10.
Abstract: Polymerase-mediated single-base extension
(SBE) of primers using a fluorescently labeled
20,30-dideoxynucleotide triphosphate terminator was originally
commercialized as SNaPshot for analysis of single-
nucleotide polymorphisms by capillary
electrophoresis (CE). Application of this general method
to bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically
infer polymorphic methylation status (i.e., 5-methylcytosine [5mC] vs.
cytosine [C]) in CpG-rich regions of gDNA has been noted previously by
others to be limited by CE mobility-shifted peaks for SBE products derived from
guanine (G)/ adenine (A)-mixed-base primers used to hybridize to possible
polymorphic sites (i.e., C vs. thymine [T] resulting from 5mC vs. C,
respectively). This limitation is precluded in the current study by using
novel SNaPshot primers modified with N6-methoxy-2,6-diaminopurine (K),
which was originally described in 1991 by Brown and Lin as a unique
adenine–guanine analog capable of participating in three H-bonds with C or
T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are
commercially available or can be readily synthesized, and they may have
utility in other assay formats used to analyze CpG methylation status.
Abstract: This protocol describes a single nucleotide
polymorphism (SNP) genotyping strategy for highly degraded DNA, using a
two-stage multiplex whereby multiple fragments are first amplified in a
single exponential reaction and the products of this PCR are added to
a linear single-base-extension reaction. It utilizes the analytical power
of a capillary electrophoresis system to simultaneously type all the
target sites. The protocol is specifically written for use with severely
fragmented templates, typical of ancient DNA, and can be adapted to widely
used detection platforms. The addition of the single-phase genotyping step
avoids the need for the re-amplification and cloning of PCR products,
while providing its own controls for the detection of contamination and allelic
drop-out. This protocol can facilitate the routine analysis of up to 52
SNP markers (haploid or diploid) in 96 samples in a single day, and
is recommended for the authentication of data in all areas of DNA research
(population and medical genetics, forensics, ancient DNA).
Abstract: The aim of the present study was to
investigate the use of the SNaPshot minisequencing method for
the identification of Mycobacterium tuberculosis complex (MTBC) isolates
to the species level and for further genotyping of M. tuberculosis
isolates. We developed an innovative strategy based on two multiplex
allelespecific minisequencing assays that allowed detection of eight
species-specific and eight lineage-specific single nucleotide
polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification,
followed by an eightplex minisequencing reaction with the SNaPshot
multiplex kit (Applied Biosystems) and, finally, analysis of the extension
products by capillary electrophoresis. The whole strategy was developed with a
panel of 56 MTBC strains and 15 negative controls. All MTBC strains tested
except one M. africanum clinical isolate were accurately identified to the
species level, and all M. tuberculosis isolates were successfully further
genotyped. This two-step strategy based on SNaPshot minisequencing allows
the simultaneous differentiation of closely related members of the MTBC,
the distinction between principal genetic groups, and the characterization
of M. tuberculosis isolates into one of the seven prominent SNP cluster
groups (SCGs) and could be a useful tool for diagnostic and
epidemiological purposes.
Background
In the workup of patients with suspected hereditary
nonpolyposis colorectal cancer (HNPCC), detection of loss of
heterozygosity (LOH) could help pinpoint the mismatch-repair (MMR) gene
carrying the germline mutation, but analysis of
microsatellite markers has proved unreliable for this purpose.
We developed a simple, low-cost method based on
singlenucleotide polymorphism (SNP) genotyping and
capillary electrophoresis for the assessment of LOH at 2 MMR loci
simultaneously.
Methods
We used the Applied Biosystems SNaPshot® Multiplex Kit with
meticulously selected primers to assess 14 common SNPs in MLH1 [mutL
homolog 1, colon cancer, nonpolyposis type 2 (E. coli)] and
MSH2 [mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coli)]
and optimized the protocol for DNA isolated from peripheral blood and
fresh/frozen or archival microsatellite-unstable tumors from patients with
confirmed (n42) or suspected (n25) HNPCC. The 42 tumors from patients
with confirmed MLH1 or MSH2 germline mutations were used to validate the
method’s diagnostic accuracy against results obtained with
DNA sequencing or multiplex ligation-dependent probe amplification.
Abstract: The development and use of high
throughput technologies for detailed mapping of methylated
cytosines (metC) is becoming of increasing importance for
the expanding field of epigenetics. The single nucleotide primer
extension reaction used for genotyping of single nucleotide polymorphisms
has been recently adapted to interrogate the bisulfite
modification induced ‘quantitative’ C/T polymorphism that
corresponds to metC/C in the native DNA. In this study, we explored
the opportunity to investigate C/T (and G/A) ratios using the Applied
Biosystems (ABI) SNaPshot technology. The main effort of this study was
dedicated to addressing the complexities in the analysis of DNA
methylation in GC-rich regions where interrogation of the target cytosine
can be confounded by variable degrees of methylation in other
cytosines (resulting in variable C/T or G/A ratios after treatment with
bisulfite) in the annealing site of the interrogating primer. In our
studies, the mismatches of the SNaPshot primer with the target DNA
sequence resulted in a biasing effect of up to 70% while
these effects decreased as the location of the polymorphic site moved
upstream of the target cytosine. We demonstrated that the biasing effect
can be corrected with the SNaPshot primers containing
degenerative C/T and G/A nucleotides. A series of experiments using
various permutations of quantitative C/T and G/A polymorphisms at various
positions of the target DNA sequence demonstrated that SNaPshot is
able to accurately report cytosine methylation levels with <5% average
SD from the true values. Given the relative simplicity of the method and
the possibility to multiplex C/T and G/A interrogations, the
SNaPshot approach may become a useful tool for large-scale mapping of
metC.
Abstract: The tiger (Panthera tigris) is
currently listed on Appendix I of the Convention on the International Trade
in Endangered Species of Wild Fauna and Flora; this affords it the highest
level of international protection. To aid in the investigation of alleged
illegal trade in tiger body parts and derivatives, molecular approaches
have been developed to identify biological material as being of tiger in
origin. Some countries also require knowledge of the exact tiger
subspecies present in order to prosecute anyone alleged to be trading in
tiger products. In this study we aimed to develop and validate a reliable
single assay to identify tiger species and subspecies simultaneously; this
test is based on identification of single nucleotide polymorphisms (SNPs)
within the tiger mitochondrial genome. The mitochondrial DNA sequence
from four of the five extant putative tiger subspecies that currently
exist in the wild were obtained and combined with DNA sequence data from
492 tiger and 349 other mammalian species available on GenBank. From the
sequence data a total of 11 SNP loci were identified as suitable for further
analyses. Five SNPs were species-specific for tiger and six amplify one of
the tiger subspecies-specific SNPs, three of which were specific to P. t.
sumatrae and the other three were specific to P. t. tigris. The multiplex
assay was able to reliably identify 15 voucher tiger samples. The
sensitivity of the test was 15,000 mitochondrial DNA copies (approximately
0.26 pg), indicating that it will work on trace amounts of tissue, bone or
hair samples. This simple test will add to the DNA-based methods currently
being used to identify the presence of tiger within mixed samples.
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