High-throughput AFLP analysis using infrared dye-labeled primers and an automated DNA sequencer.
نویسندگان
چکیده
Amplified fragment length polymorphism (AFLP) analysis is currently the most powerful and efficient technique for the generation of large numbers of anonymous DNA markers in plant and animal genomes. We have developed a protocol for high-throughput AFLP analysis that allows up to 70,000 polymorphic marker genotype determinations per week on a single automated DNA sequencer. This throughput is based on multiplexed PCR amplification of AFLP fragments using two different infrared dyelabeled primer combinations. The multiplexed AFLPs are resolved on a two-dye, model 4200 LI-COR automated DNA sequencer, and the digital images are scored using semi-automated scoring software specifically designed for complex AFLP banding patterns (AFLP-Quantar). Throughput is enhanced by using high-quality genomic DNA templates obtained by a 96-well DNA isolation procedure.
منابع مشابه
Automated recording of RNA differential display patterns from pig granulosa cells.
We have developed a protocol for fast, nonradioactive, mRNA differential display reverse transcription PCR (DDRT-PCR) based on a commercial automated sequencer with RNA isolated from pig granulosa cells. We sought to discover conditions that would minimize the problem of using relatively small primers labeled with large infrared dye molecule, IR41, required for the sequencer. Extended IR41-labe...
متن کاملGeneration of multiple mRNA fingerprints using fluorescence-based differential display and an automated DNA sequencer.
Differential display is a method for the survey, analysis and comparison of gene expression in eukaryotic cells and tissues. Differential display involves isolation of high-quality nondegraded RNA, selective reverse transcription of polyadenylated mRNA using specific anchored oligopolydeoxythymidine [oligo(dT)] primers, and the subsequent PCR amplification of the cDNA with the same oligo(dT), a...
متن کاملInfrared fluorescent detection of D1S80 alleles from blood and body fluid collected on IsoCode devices.
A genetic locus D1S80 (pMCT 118) containing variable number of tandem repeats (VNTR) has been used extensively in forensic analysis and paternity testing. In the current research, DNA was isolated from blood saliva and nasal secretions collected on two types of IsoCode paper-based devices. The D1S80 locus was amplified using PCR technology, and the alleles were separated by gel electrophoresis ...
متن کاملAn improved, high-throughput method for detection of bluetongue virus RNA in Culicoides midges utilizing infrared-dye-labeled primers for reverse transcriptase PCR.
A new rapid (less than 6h from insect-to-results) high-throughput assay that is sensitive and specific for detecting BTV RNA in Culicoides biting midges is reported. Homogenization and extraction of nucleic acids from individual Culicoides specimens were performed in a 96-well plate format using specialized beads in a homogenization buffer compatible with cell culture and RNA extraction. A port...
متن کاملElectrophoretic mobility shift scanning using an automated infrared DNA sequencer.
Electrophoretic mobility shift assay (EMSA) is widely used in the study of sequence-specific DNA-binding proteins, including transcription factors and mismatch binding proteins. We have established a non-radioisotope-based protocol for EMSA that features an automated DNA sequencer with an infrared fluorescent dye (IRDye) detection unit. Our modification of the elec- trophoresis unit, which incl...
متن کاملذخیره در منابع من
با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید
برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید
ثبت ناماگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید
ورودعنوان ژورنال:
- BioTechniques
دوره 30 2 شماره
صفحات -
تاریخ انتشار 2001