ISSR (Inter-Simple Sequence Repeat) Analysis
Inter-simple sequence repeat (ISSR) PCR is a fast, inexpensive genotyping technique based on variation in the regions between microsatellites. This method has a wide range of uses, including the characterization of genetic relatedness among populations, genetic fingerprinting, gene tagging, detection of clonal variation, cultivar identification, phylogenetic analysis, detection of genomic instability, and assessment of hybridization.
Figure 1: ISSR Genotyping
ISSR genotyping of endangered plants using an optimized workflow (Figure 1). Applied Biosystems, Inc. (2010)
Experimental Workflow: Find the Products You Need for Every Step
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Step 1: DNA Purification
DNA extraction is a critical first step in the experimental workflow of DNA Sequencing and Fragment analysis. The overall quality, accuracy and length of the DNA sequence read can be significantly affected by characteristics of the sample itself, and the method chosen for nucleic acid extraction.
Ideal methods will vary depending on the source or tissue type, how it was obtained from its source, and how the sample was handled or stored prior to extraction.
Step 2: Primer Design & PCR Amplification
Amplify inter-simple sequence repeat loci of interest with a fluorescently labeled primer directed against di- or tri-nucleotide repeats
Products for Step 2: Primer Design & PCR Amplification
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AmpliTaq Gold® 360 Master Mix
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AmpliTaq Gold® 360 PCR Master Mix contains everything required for successful PCR amplification in one convenient package, with all components premixed and premeasured. AmpliTaq Gold 360 Master Mix was designed for 360° coverage of a full range of targets.
Step 3: Sample Prep for Fragment Analysis Run
Add aliquot of PCR reaction to size standard and Hi-Di Formamide mix; heat denature and proceed with electrophoresis.
Products for Step 3: Sample Prep for Fragment Analysis Run
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Hi-Di™ Formamide
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Highly deionized formamide used to resuspend samples before electrokinetic injection in capillary electrophoresis systems.
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GeneScan™ Size Standards
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GeneScan™ Size Standards are fluorescently-labeled DNA fragments required for performing fragment analysis and genotyping applications on our capillary electrophoresis platforms.
Step 4: Capillary Electrophoresis
During capillary electrophoresis, the products of the PCR reaction are injected electrokinetically into capillaries filled with polymer. High voltage is applied so that the negatively charged DNA fragments move through the polymer in the capillaries toward the positive electrode.
Products for Step 4: Capillary Electrophoresis
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3500 Genetic Analyzer
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The 3500 8-capillary platform can run a wide variety of applications—including de novo sequencing and resequencing (mutational profiling)—as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.
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3500xl Genetic Analyzer
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The 3500xL 24-capillary platform can run a wide variety of applications, including de novo sequencing and resequencing (mutational profiling), as well as microsatellite analysis, MLPA™, LOH, MLST, AFLP® and SNP validation or screening. The majority of applications can be run on a single polymer and capillary array.
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3730 DNA Analyzer
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The 48-capillary 3730 DNA Analyzer is the Gold Standard in medium-to-high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample. This is upgradeable to 96 capillaries.
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3730xl DNA Analyzer
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The 96-capillary 3730xl DNA Analyzer is the Gold Standard for high throughput genetic analysis. Use this for DNA fragment analysis applications such as microsatellites, AFLP, SNP analysis, mutation detection and traditional DNA sequencing. Get the highest quality data at a low cost per sample.
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3130 Genetic Analyzer
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Applied Biosystems 3130 Genetic Analyzer is the latest generation of 4-capillary electrophoresis instruments for the low to medium throughput laboratories. The system offers industry-leading performance, plus sophisticated automation capabilities allowing you to save time, reduce costs and increase productivity.
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3130xl Genetic Analyzer
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Applied Biosystems 3130xl Genetic Analyzer is the latest generation of 16-capillary electrophoresis instruments for the medium throughput laboratories.
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310 Genetic Analyzer
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The ABI PRISM® 310 Genetic Analyzer is an automated single-capillary genetic analyzer designed for a wide range of sequencing and fragment analysis applications.
Step 5: Data Analysis
Size fragments, score alleles, assess replicate concordance and build phylograms.
Products for Step 5: Data Analysis
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GeneMapper® Software
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GeneMapper is a flexible genotyping software package that provides DNA sizing and quality allele calls for all Applied Biosystems electrophoresis-based genotyping systems.
