Aplicações da Técnica de SNaPshot
Autor: Ricardo Dalla Costa | Publicado: 2012-09-23T20:22:00-07:00


A técnica de SNaPshot® é baseada na incorporação de um único nucleotide marcado com fluorescência (ddNTP) a um fragmento de DNA não marcado. Após a reação de SNaPshot o produto é misturado com formamida e um size standard e submetido á eletroforese capilar para identificação dos fragmentos marcados com fluorescência. Através desta técnica, é possível genotipar SNPs (Figura 1), identificar regiões metiladas, mapear BACs, entre outras aplicações. Veja abaixo alguns artigos com o uso dessa técnica.



Figura 1. SNaPshot® labeling chemistry relies on single-base extension and termination.&nbsp;The SNaPshot® Multiplex Kit uses a single-tube reaction to interrogate SNPs at known&nbsp;locations. The chemistry is based on the dideoxy single-base extension of an unlabeled&nbsp;oligonucleotide primer (or primers). Each primer binds to a complementary template in the&nbsp;presence of fluorescently labeled ddNTPs and DNA polymerase. The polymerase extends&nbsp;the primer by one nucleotide, adding a single ddNTP to its 3´ end. The fluorescence color&nbsp;readout reports which base was added.

SNaPshot® Multiplex System for SNP genotyping.pdf
Veja nesse Application Note uma lista de vários artigos que usaram a técnica de SNaPshot para diferentes finalidades.

Abstract:&nbsp;The SNaPshot® Multiplex System is&nbsp;a primer extension–based method&nbsp;developed for the analysis of single&nbsp;nucleotide polymorphisms (SNPs)&nbsp;(Figure 1). Through its multiplexing&nbsp;capability, up to 10 SNPs can be analyzed&nbsp;in a single reaction, regardless&nbsp;of their positions on the chromosome&nbsp;or the amount of separation from&nbsp;neighboring SNP loci. The ability to&nbsp;use unlabeled, user-defined primers&nbsp;allows researchers to incorporate&nbsp;SNPs of interest cost-effectively.&nbsp;The Multiplex Ready Reaction Mix&nbsp;(included in the system) helps ensure&nbsp;robust, reproducible analyses of&nbsp;multiplexed samples. Researchers&nbsp;can analyze more than 23,000 SNP&nbsp;genotypes per day on just one 3730xl&nbsp;Genetic Analyzer.


Generated BAC Fingerprinting on Capillary Electrophoresis System.pdf

White paper que descreve o uso da técnica de Análise de Fragmentos para o mapeamento gênico

Abstract: BAC fingerprinting provides an efficient&nbsp;and cost-effective method of characterizing&nbsp;large genomic fragment libraries for&nbsp;genome sequencing, positional cloning,&nbsp;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.


Capillary electrophoretic analysis of methylation status in CpG-rich regions by single-base extension of primers modified with N6-methoxy-2,6-diaminopurine.pdf

Abstract:&nbsp;Polymerase-mediated single-base extension (SBE) of primers using a fluorescently labeled 20,30-dideoxynucleotide&nbsp;triphosphate terminator was originally commercialized as SNaPshot for analysis of single-

nucleotide polymorphisms by capillary electrophoresis (CE). Application of this general method to&nbsp;bisulfite-converted/PCR-amplified genomic DNA (gDNA) to analytically infer polymorphic methylation&nbsp;status (i.e., 5-methylcytosine [5mC] vs. cytosine [C]) in CpG-rich regions of gDNA has been noted previously&nbsp;by others to be limited by CE mobility-shifted peaks for SBE products derived from guanine (G)/&nbsp;adenine (A)-mixed-base primers used to hybridize to possible polymorphic sites (i.e., C vs. thymine [T]&nbsp;resulting from 5mC vs. C, respectively). This limitation is precluded in the current study by using novel&nbsp;SNaPshot primers modified with N6-methoxy-2,6-diaminopurine (K), which was originally described in&nbsp;1991 by Brown and Lin as a unique adenine–guanine analog capable of participating in three H-bonds&nbsp;with C or T in DNA. Oligonucleotides modified by K as a bispecific complement for C/T are commercially&nbsp;available or can be readily synthesized, and they may have utility in other assay formats used to analyze&nbsp;CpG methylation status.



Developing multiplexed SNP assays with special reference to degraded DNA templates.pdf

Abstract:&nbsp;This protocol describes a single nucleotide polymorphism (SNP) genotyping strategy for highly degraded DNA, using a two-stage&nbsp;multiplex whereby multiple fragments are first amplified in a single exponential reaction and the products of this PCR are added to a&nbsp;linear single-base-extension reaction. It utilizes the analytical power of a capillary electrophoresis system to simultaneously type&nbsp;all the target sites. The protocol is specifically written for use with severely fragmented templates, typical of ancient DNA, and&nbsp;can be adapted to widely used detection platforms. The addition of the single-phase genotyping step avoids the need for the&nbsp;re-amplification and cloning of PCR products, while providing its own controls for the detection of contamination and allelic drop-out.&nbsp;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&nbsp;recommended for the authentication of data in all areas of DNA research (population and medical genetics, forensics, ancient DNA).



Identification and Genotyping of Mycobacterium tuberculosis Complex Species by Use of a SNaPshot Minisequencing-Based Assay.pdf

Abstract:&nbsp;The aim of the present study was to investigate the use of the SNaPshot minisequencing method for the&nbsp;identification of Mycobacterium tuberculosis complex (MTBC) isolates to the species level and for further&nbsp;genotyping of M. tuberculosis isolates. We developed an innovative strategy based on two multiplex allelespecific&nbsp;minisequencing assays that allowed detection of eight species-specific and eight lineage-specific single&nbsp;nucleotide polymorphisms (SNPs). Each assay consisted of an eightplex PCR amplification, followed by an&nbsp;eightplex minisequencing reaction with the SNaPshot multiplex kit (Applied Biosystems) and, finally, analysis&nbsp;of the extension products by capillary electrophoresis. The whole strategy was developed with a panel of 56&nbsp;MTBC strains and 15 negative controls. All MTBC strains tested except one M. africanum clinical isolate were&nbsp;accurately identified to the species level, and all M. tuberculosis isolates were successfully further genotyped.&nbsp;This two-step strategy based on SNaPshot minisequencing allows the simultaneous differentiation of closely&nbsp;related members of the MTBC, the distinction between principal genetic groups, and the characterization of&nbsp;M. tuberculosis isolates into one of the seven prominent SNP cluster groups (SCGs) and could be a useful tool&nbsp;for diagnostic and epidemiological purposes.



Multiplex SNaPshot Genotyping for Detecting Loss of Heterozigozity.pdf

Background

In the workup of patients with suspected&nbsp;hereditary nonpolyposis colorectal cancer (HNPCC),&nbsp;detection of loss of heterozygosity (LOH) could help&nbsp;pinpoint the mismatch-repair (MMR) gene carrying&nbsp;the germline mutation, but analysis of microsatellite&nbsp;markers has proved unreliable for this purpose. We&nbsp;developed a simple, low-cost method based on singlenucleotide&nbsp;polymorphism (SNP) genotyping and capillary&nbsp;electrophoresis for the assessment of LOH at 2&nbsp;MMR loci simultaneously.&nbsp;

Methods

We used the Applied Biosystems SNaPshot®&nbsp;Multiplex Kit with meticulously selected primers to assess&nbsp;14 common SNPs in MLH1 [mutL homolog 1,&nbsp;colon cancer, nonpolyposis type 2 (E. coli)] and MSH2&nbsp;[mutS homolog 2, colon cancer, nonpolyposis type 1&nbsp;(E. coli)] and optimized the protocol for DNA isolated&nbsp;from peripheral blood and fresh/frozen or archival microsatellite-unstable tumors from patients with confirmed&nbsp;(n42) or suspected (n25) HNPCC. The 42&nbsp;tumors from patients with confirmed MLH1 or MSH2&nbsp;germline mutations were used to validate the method’s&nbsp;diagnostic accuracy against results obtained with DNA&nbsp;sequencing or multiplex ligation-dependent probe&nbsp;amplification.



Single nucleotide extension technology for quantitative site-specific evaluation of metC-C in GC-rich regions.pdf

Abstract:&nbsp;The development and use of high throughput technologies&nbsp;for detailed mapping of methylated cytosines&nbsp;(metC) is becoming of increasing importance for the&nbsp;expanding field of epigenetics. The single nucleotide&nbsp;primer extension reaction used for genotyping of&nbsp;single nucleotide polymorphisms has been recently&nbsp;adapted to interrogate the bisulfite modification&nbsp;induced ‘quantitative’ C/T polymorphism that corresponds&nbsp;to metC/C in the native DNA. In this study, we&nbsp;explored the opportunity to investigate C/T (and G/A)&nbsp;ratios using the Applied Biosystems (ABI) SNaPshot&nbsp;technology. The main effort of this study was dedicated&nbsp;to addressing the complexities in the analysis&nbsp;of DNA methylation in GC-rich regions where interrogation&nbsp;of the target cytosine can be confounded&nbsp;by variable degrees of methylation in other cytosines&nbsp;(resulting in variable C/T or G/A ratios after treatment&nbsp;with bisulfite) in the annealing site of the interrogating&nbsp;primer. In our studies, the mismatches of the&nbsp;SNaPshot primer with the target DNA sequence resulted&nbsp;in a biasing effect of up to 70% while these&nbsp;effects decreased as the location of the polymorphic&nbsp;site moved upstream of the target cytosine. We&nbsp;demonstrated that the biasing effect can be corrected&nbsp;with the SNaPshot primers containing degenerative&nbsp;C/T and G/A nucleotides. A series of experiments&nbsp;using various permutations of quantitative C/T and&nbsp;G/A polymorphisms at various positions of the target&nbsp;DNA sequence demonstrated that SNaPshot is able&nbsp;to accurately report cytosine methylation levels with &lt;5% average SD from the true values. Given the relative&nbsp;simplicity of the method and the possibility to&nbsp;multiplex C/T and G/A interrogations, the SNaPshot&nbsp;approach may become a useful tool for large-scale&nbsp;mapping of metC.



The development and validation of a single SNaPshot multiplex for tiger species and subspecies identification-Implications for forensic purposes.pdf

Abstract:&nbsp;The tiger (Panthera tigris) is currently listed on Appendix I of the Convention on the International Trade in&nbsp;Endangered Species of Wild Fauna and Flora; this affords it the highest level of international protection.&nbsp;To aid in the investigation of alleged illegal trade in tiger body parts and derivatives, molecular&nbsp;approaches have been developed to identify biological material as being of tiger in origin. Some countries&nbsp;also require knowledge of the exact tiger subspecies present in order to prosecute anyone alleged to be&nbsp;trading in tiger products. In this study we aimed to develop and validate a reliable single assay to identify&nbsp;tiger species and subspecies simultaneously; this test is based on identification of single nucleotide&nbsp;polymorphisms (SNPs) within the tiger mitochondrial genome. The mitochondrial DNA sequence from&nbsp;four of the five extant putative tiger subspecies that currently exist in the wild were obtained and&nbsp;combined with DNA sequence data from 492 tiger and 349 other mammalian species available on&nbsp;GenBank. From the sequence data a total of 11 SNP loci were identified as suitable for further analyses.&nbsp;Five SNPs were species-specific for tiger and six amplify one of the tiger subspecies-specific SNPs, three&nbsp;of which were specific to P. t. sumatrae and the other three were specific to P. t. tigris. The multiplex assay&nbsp;was able to reliably identify 15 voucher tiger samples. The sensitivity of the test was 15,000&nbsp;mitochondrial DNA copies (approximately 0.26 pg), indicating that it will work on trace amounts of&nbsp;tissue, bone or hair samples. This simple test will add to the DNA-based methods currently being used to&nbsp;identify the presence of tiger within mixed samples.