Microsatellites: Methods and Protocols (Methods in Molecular Biology, 1006)

✍ Scribed by Stella K. Kantartzi (editor)

• Publisher: Humana
• Year: 2013
• Tongue: English
• Leaves: 337
• Category: Library

✦ Synopsis

Microsatellites or simple sequence repeats (SSRs) have become the markers of choice for a variety of molecular studies because of their versatility, operational flexibility, and lower cost than other marker systems.  Microsatellites: Methods and Protocols brings together experts in the field to cover this significant area of research.  Broken in to four convenient parts, this volume delves into classical and modern methods for the discovery and development of microsatellite markers, descriptions of amplification and visualization of SSRs, automated capillary sequencers that are widely used for fragment analysis, as well as a variety of methods for the analysis of data obtained by the use of microsatellites. Written for the highly successful Methods in Molecular Biology™ series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.

 

Authoritative and invaluable, Microsatellites: Methods and Protocols aims at researchers that need detailed protocols for incorporating microsatellite markers into their projects and expert scientists looking to expand their knowledge of SSRs discovery, use, and analysis.

✦ Table of Contents

Microsatellites
Preface
Contents
Contributors
Chapter 1: Microsatellites: Evolution and Contribution
1 Introduction
2 Microsatellites
3 Generation of Microsatellite Diversity
4 Replication Slippage
5 Recombination
6 Infrastructure and Methods for the Study of Microsatellites
7 Technical Problems and Difficulties in Studying Microsatellites
8 Advances in Microsatellites
9 The Advances of High-Resolution Melting Analysis in Microsatellite Studies
10 Applications of Microsatellites
11 Conclusions
References
Part I: Discovery and Development of Microsatellites
Chapter 2: Screening of Genomic Libraries
1 Introduction
2 Materials and Methods
2.1 DNA Extraction
2.2 Genomic DNA Digestion
2.3 Gel-Fractionate to Isolate DNA Fragments
2.4 Ligation of Adapters
2.5 Biotin-Labeled Oligonucleotide
2.6 Preparation of Magnetic Beads
2.7 Hybridization
2.8 PCR Control for Enrichment Procedure and Chemiluminescent Probe Detection
2.9 Hybridization
2.10 Cloning of PCR Fragments
References
Chapter 3: PCR-Based Isolation of Microsatellite Arrays (PIMA)
1 Introduction
2 Materials (Table  2)
2.1 PCR-Based Identification of Microsatellite Arrays
2.2 Preparation for Genomic DNA of Target Species
2.3 Preparation of Random Amplified Polymorphic DNA Primers
2.4 PCR-Based Identification of Microsatellite Arrays Experiments
2.4.1 Preparation for Cloning Experiments
2.4.2 Preparation for Positive Clone Screening by PCR
2.4.3 Microsatellite Detection
2.4.4 Colony Sequencing
2.4.5 Primer Design
3 Methods
3.1 Genomic DNA Preparation
3.1.1 Cetyltrime-thylammonium Bromide Method ( 16)
3.1.2 DNeasy Plant Mini Kit Method
3.2 Random Amplified Polymorphic DNA Fragment Construction
3.3 Clone Operation and Screening
3.3.1 Ligation Using the pGEM-T and pGEM-T Easy Vector (Promega)
3.3.2 Transformation Experiment
3.3.3 Transformation Efficiency Calculation
3.3.4 Screening the Transformants for Inserts
3.4 Microsatellite Detection
3.5 Data Analysis
4 Notes
References
Chapter 4: Fast Isolation by AFLP of Sequences Containing Repeats
1 Introduction
2 Materials
2.1 Digestion–Ligation Reagents
2.2 PCR Amplification Reagents
2.3 Hybridization and Washing Reagents
2.4 Other Reagents and Equipments
3 Methods
3.1 Digestion–Ligation
3.2 Optimization of Amplification
3.3 Hybridization and Harvesting
3.4 Amplification of the Enriched Fragments
4 Notes
References
Chapter 5: Microsatellite DNA Capture from Enriched Libraries
1 Introduction
2 Materials
2.1 Microsatellite Isolation
2.1.1 Microsatellite Enrichment and Library Construction
2.1.2 Screening and Isolation of Positive Clones Containing Microsatellites
2.2 Next-Generation Sequencing Methods for Microsatellite Capture
2.3 PCR Primer Design, Testing, and Basic Genotyping
3 Methods
3.1 Microsatellite Isolation
3.1.1 Microsatellite Enrichment and Library Construction
3.1.2 Screening, Isolation, and Sequencing of Clones Containing Microsatellites
3.2 Next-Generation Sequencing Methods for Microsatellite Capture
3.3 PCR Primer Design, Testing, and Basic Genotyping
4 Notes
References
Chapter 6: Next-Generation Sequencing for High-Throughput Molecular Ecology: A Step-by-Step Protocol for Targeted Multilocu...
1 Introduction
2 Materials
2.1 Experimental Design and Primer Barcoding
2.2 Locus Amplification and Purification Materials
2.3 Quantification and Pooling Materials
2.4 Data Analysis Materials
3 Methods
3.1 Experimental Design and Primer Barcoding
3.2 Locus Amplification and Purification
3.3 Quantification and Pooling
3.4 Data Analysis
4 Notes
References
Chapter 7: Optimizing Selection of Microsatellite Loci from 454 Pyrosequencing via Post-sequencing Bioinformatic Analyses
1 Introduction
1.1 File Conversion from SFF to FASTQ with sff_extract ( 16)
1.2 Library Splitting by Barcode Matching Using the barcode_splitter Script from the Fastx_toolkit ( 17)
1.3 Evaluation of the Quality of the Sequences in the Raw Data (Before Quality Control) with FastQC ( 18)
1.4 Quality Control, Elimination of Short Reads, and Barcode Clipping with clean_reads ( 16)
1.5 Evaluation of the Quality of the Sequences in the Clean Data (After Quality Control) with FASTQC
1.6 Detection of Microsatellite-Containing Sequences Using the pipe1.pl Script of QDD ( 19)
1.7 Similarity Analysis Using the pipe2.pl Script of QDD
1.8 Avoiding Sequences with Homology to Repetitive Elements Using Censor ( 20)
1.9 Design of Primers Flanking Microsatellite Repeats
1.10 Similarity Analysis with BLASTALL to Avoid Using Duplicated Sequences for the Design of Microsatellites
2 Software
3 Methods
3.1 File Conversion from SFF to FASTQ with sff_extract
3.2 Library Splitting by Barcode Matching Using barcode_splitter from the Fastx_toolkit
3.3 Evaluation of the Quality of the Sequences in the Raw Data (Before Quality Control) with FastQC
3.4 Quality Control, Elimination of Short Reads, and Barcode Clipping with clean_reads
3.5 Evaluation of the Quality of the Sequences in the Clean Data (After Quality Control) with FastQC
3.6 Detection of Microsatellite-Containing Sequences Using the pipe1.pl Script of QDD
3.7 Similarity Analysis Using the pipe2.pl Script of QDD
3.8 Avoiding Sequences with Homology to Repetitive Elements Using Censor
3.9 Design of Primers Flanking Microsatellite Repeats
3.10 Similarity Analysis with BLASTALL to Avoid Using Duplicated Sequences for the Design of Microsatellites
4 Notes
References
Chapter 8: Identification of DNA-Microsatellite Markers for the Characterization of Somatic Embryos in Quercus suber
1 Introduction
2 Materials
2.1 Solutions for DNA Extraction from Embryos
2.2 Solutions for DNA Extraction from Leaves
2.3 Buffer for DNA Quantification
2.4 Chemicals for SSR Amplifications
2.5 Equipment
2.6 Plastic Ware ( See Note 3)
3 Methods
3.1 DNA Extraction from Embryos
3.2 DNA Extraction from Leaves
3.3 DNA Quantification
3.4 SSR Amplifications by PCR
3.5 Analyzing the Data
4 Notes
References
Part II: Amplification and Visualization
Chapter 9: Simple Sequence Repeats Amplification
1 Introduction
2 Materials
3 Methods
3.1 Amplification Using Unlabeled SSR Primers
3.2 Amplification Using Labeled SSR Primers
3.3 Amplification Using M13-Tailed SSR Primers and M13-Labeled Probes
3.4 Multiplex PCR
4 Notes
References
Chapter 10: Microsatellite Amplification in Plants: Optimization Procedure of Major PCR Components
1 Introduction
2 Materials
3 Methods
3.1 Optimize Reaction Components
3.1.1 Optimize Amount of MgCl 2
3.1.2 Optimize Amount of Taq DNA Polymerase
3.1.3 Optimize Amount of Microsatellite Primer
3.1.4 Optimize Amount of Template DNA
3.1.5 Checking of PCR Amplification
4 Notes
References
Chapter 11: Development of a Multiplex PCR Assay for Characterization of Embryonic Stem Cells
1 Introduction
1.1 Need for Alternate Methods Toward Simple and Cost-Effective Characterization of hESC
1.2 How Can Multiplex PCR Become a Suitable Screening Tool?
1.3 Specific Advantages and Limitations of This Method
1.3.1 Indication of Template Quality
1.3.2 Indication of Template Quantity
1.3.3 Internal Controls
1.3.4 Use as a Molecular Weight Ladder
1.3.5 Efficiency
1.3.6 Limitations
2 Materials
2.1 Equipments, Consumables, and Cell Lines
2.2 Culturing Human ES Cells
2.3 RNA Isolation and cDNA Synthesis
2.4 PCR and Primers/Oligos
3 Methods
3.1 Culture and Propagation of Human ES Cell Lines
3.2 Differentiation Induction by EB Formation
3.3 Total RNA Extraction and cDNA Synthesis
3.4 Standardizing Independent Sets of Multiplex PCR and in Combinations
3.5 Validation of Multiplex PCR
4 Notes
4.1 Precautions to Be Taken
4.1.1 Positioning of Primers
4.1.2 Developing Primers and Reaction Conditions
4.1.3 Titration of Reaction Components
4.1.4 Selection of Multiplex Loci
4.1.5 Adapting Thermocycling Conditions
4.1.6 Competition and Interference
4.1.7 Post-PCR Analysis
4.2 Detection Sensitivity Compared to Other Similar Methods
4.3 Problems Encountered: Primer Interface, Cross-Reactivity, and Cycling Conditions
4.4 Other Molecular Biology Applications of Multiplex PCR
4.4.1 Gene Deletion and Mutation Detection
4.4.2 Polymorphic Repetitive DNA
4.4.3 Microbe Detection and Characterization
References
Chapter 12: Agarose Gel Electrophoresis and Polyacrylamide Gel Electrophoresis for Visualization of Simple Sequence Repeats
1 Introduction
2 Materials
2.1 Agarose Gel Electrophoresis
2.2 Polyacrylamide Gel Electrophoresis
3 Methods
3.1 Agarose Gel
3.2 Polyacrylamide Gel
References
Part III: Automated Capillary Sequencers
Chapter 13: Microsatellite Fragment Analysis Using the ABI Prism ® 377 DNA Sequencer
1 Introduction
2 Materials
2.1 Polyacrylamide Gel Casting
2.2 Electrophoresis of PCR-Amplified Fragments
3 Methods
3.1 Polyacrylamide Gel Casting
3.2 Electrophoresis of PCR-Amplified Fragments
3.3 Sizing and Scoring Microsatellite Fragments ( See Note 8)
3.4 Troubleshooting
4 Notes
References
Chapter 14: Robust and Inexpensive SSR Markers Analyses Using LI-COR DNA Analyzer
1 Introduction
2 Materials
2.1 Total DNA
2.2 Reagents and Components for SSR Marker Amplification by PCR
2.3 Reagents and Components for PAGE on LI-COR Apparatus
3 Methods
3.1 SSR Marker Amplification by PCR
3.2 PAGE Preparation
3.3 Detection of Amplicons
4 Notes
References
Chapter 15: The Use of the MegaBACE for Sequencing and Genotype Analysis
1 Introduction
2 Materials
2.1 Quantification of PCR Products
2.2 Sequencing Reaction
2.3 Purification of Sequencing Products
2.4 Genotyping with Microsatellite Loci
2.5 Running the MegaBACE
3 Methods
3.1 Quantification of PCR Products
3.2 Sequencing Reaction
3.3 Purification of Sequencing Products
3.3.1 Purification of Sequencing Products by Gel Filtration
3.3.2 Purification of Sequencing Products by Salt/Ethanol Precipitation
3.4 Genotyping with Microsatellite Loci
3.4.1 Multiplex PCR Reaction Using Fluorescent-Labeled Microsatellite Primers
3.4.2 Dilution of the PCR Product
3.5 Running the MegaBACE
3.5.1 Switch on the MegaBACE
3.5.2 Before Starting a Run
3.5.3 Heating Up the MegaBACE
3.5.4 Rinse Tips
3.5.5 Start the MegaBACE Run
3.5.6 Inject Samples
3.5.7 Stop a MegaBACE Run
3.5.8 Store Capillaries (or Put the MegaBACE to Sleep)
3.5.9 Switch Off the MegaBACE
4 Notes
References
Chapter 16: Analyzing Microsatellites Using the QIAxcel System
1 Introduction
2 Materials
3 Methods
3.1 Selecting Alignment and DNA Size Markers
3.2 Sample/Plate Preparation
3.3 Instructions to Run Samples
3.4 After the Run Is Complete
3.5 Automatically Aligning Data (Using BioCalculator™ Software)
3.6 Manual Alignment (If Data Is Not Aligned Properly After Automatic Alignment)
3.7 Applying the Selected DNA Size Marker
3.8 Exporting Data
3.9 Multiplexing Using QIAxcel System
4 Notes
References
Part IV: Scoring and Data Analysis
Chapter 17: Microsatellite Analysis of Malaria Parasites
1 Introduction
2 Materials
2.1 Equipment
2.2 Consumables
2.3 PCR
2.4 Fragment Analysis
3 Methods
3.1 P. falciparum Microsatellites
3.1.1 Oligonucleotides
3.1.2 PCR Master Mix
3.1.3 PCR Cycling Parameters
3.1.4 Fragment Analysis
3.2 P. vivax Microsatellites
3.2.1 Oligonucleotides
3.2.2 PCR Master Mix
3.2.3 PCR Cycling Parameters
3.2.4 Fragment Analysis
3.3 Data Analysis
4 Notes
References
Chapter 18: Informativeness of Microsatellite Markers
1 Introduction
1.1 Microsatellites
1.2 Information Theory
1.3 Informativeness for Genetic Markers
2 Software
2.1 R
2.2 R/qtl
2.3 Infocalc
3 Methods
3.1 Polymorphism Information Content
3.2 Gini–Simpson Index for Genotypic Frequencies
3.3 Mutual Information for Cultivar Discrimination
3.4 Marker Informativeness for Inference of Coancestry
3.5 Information for QTL Mapping
4 Notes
4.1 Recommen
References
Chapter 19: Microsatellite Data Analysis for Population Genetics
1 Introduction
2 Materials
2.1 Formatting and Data Manipulation
2.2 Basic Population Genetic Analyses
2.3 Advanced Population Genetic Analyses
3 Methods
3.1 Input File and Correct File Extension for Each Program
3.2 Converting Genotype File in Excel to Txt File Format
3.3 Formatting and Data Manipulation
3.3.1 Microsatellite Toolkit
3.3.2 Micro-Checker
3.3.3 GenAlEx
3.4 Basic Population Genetic Analyses
3.4.1 Indices of Genetic Diversity
3.4.2 Test of Hardy–Weinberg Equilibrium
3.4.3 Test for Genotypic Linkage Disequilibrium
3.4.4 Measure of Fixation Indices and Genetic Differentiation Between Populations
3.4.5 Gene Flow Measures
3.4.6 Analysis of Molecular Variance Test
3.5 Advanced Population Genetic Analyses
3.5.1 Bottleneck Tests
3.5.2 Genetic Relationships Between Samples
3.5.3 Inferring Real-Time Migration Rate
3.5.4 Identification of Migrants in Current Generation
3.5.5 Assignment/Exclusion Tests
3.5.6 Inferring the Number of Distinct Genetic Populations
3.5.7 Genetic Isolation by Geographic Distance
4 Notes
Acknowledgements
References
Chapter 20: Molecular Mapping and Breeding with Microsatellite Markers
1 Introduction
2 Intellectual Property
3 Marker Development
4 Scoring of Phenotypes
5 Choice of Markers
6 Identification of Polymorphism
7 Genetic and Association Map Development
8 Marker-Assisted Recovery of Recurrent Parent Genome
9 Marker-Assisted Selection in Recurrent Cross Populations
10 Examples of Marker-Assisted Selection for Targeted Stress Resistance Traits
10.1 SCN
10.2 SDS
11 Methods for Marker-Assisted Selection
12 Conclusions
References
Chapter 21: Scoring Microsatellite Loci
1 Introduction
2 Materials
2.1 Software
3 Methods
3.1 Create a Panel
3.2 Get Familiar with Your Loci
3.3 Assess Peaks of Interest Versus PCR Artifacts
3.4 Allele Calling
3.5 Create and Export a Table
4 Notes
4.1 Previously Unreported Alleles
4.2 “Extra” Alleles
4.3 Pull-Up
4.4 Stutter in Mononucleotide and Dinucleotide Repeats
4.5 Reducing Stutter
Box 1 Improving Scoring by Reducing Stutter in Mononucleotide and Dinucleotide Repeats
4.6 Null Alleles and Allelic Drop-Out
4.7 Compound Microsatellites
4.8 Incomplete Terminal Adenylation
4.9 Controls, Confidence, and Error Rate
References
Index