By Irene Tiemann-Boege, Andrea Betancourt
This quantity info protocols for genetic, molecular, cytological, and bioinformatic equipment for identifying haplotypes. Haplotyping: equipment and Protocols publications readers via equipment that without delay variety haploid cells, difficult-to-resolve gene households, high-resolution, brief diversity haplotyping for detailed loci, and long-range haplotyping for entire chromosomes or genomes. Written within the hugely profitable Methods in Molecular Biology series structure, chapters comprise introductions to their respective issues, lists of the mandatory fabrics and reagents, step by step, without problems reproducible laboratory protocols, and tips about troubleshooting and keeping off identified pitfalls.
Authoritative and functional Haplotyping: equipment and Protocols, goals to supply researchers with an outline of experimental equipment for haplotyping.
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Extra resources for Haplotyping: Methods and Protocols
8. Centrifuge the resuspended pellet at 100 × g for 3 min at 4 °C. 28 Kyuha Choi et al. Repeat steps 7 and 8. This process should be repeated until sufficient pollen for downstream experiments has been collected. 1a). 5 ml eppendorf tubes. 1a). Pollen aliquots can be stored at −20 °C, or used directly in step 11. 11. Resuspend the pollen grain pellet in 4 vol of Lysis Buffer, containing freshly added dithiothreitol (DTT). Add proteinase K to a final concentration of 20 μg/ml. Following overnight digestion, the color of the pollen suspension will change from yellow to brown.
Carefully resuspend the pellet in 100 μl of TE buffer (see Note 2). 14. Quantify DNA concentrations using a fluorometer. 2). 15. 9) or frozen in 5–20 μl aliquots. 3 Identification of Candidate Crossover Hotspots for Pollen Typing The design and validation of hotspot allele-specific oligonucleotides (ASOs) represents a considerable amount of work, and so it is important to carefully select hotspots before the start of the experiment. A variety of methods, data and technical considerations are important in this selection procedure.
We provide detailed descriptions of key steps including pollen DNA extraction, prior identification of hotspot locations, allele-specific oligonucleotide design, and sequence analysis approaches. Together, these methods allow the rate and recombination topology of plant hotspots to be robustly measured and compared between varied genetic backgrounds and environmental conditions. Key words Meiosis, Recombination, Crossover, Allele-specific PCR, Titration, Pollen DNA 1 Introduction Meiotic recombination is conserved throughout the majority of eukaryotes, and has importance for human fertility, agricultural breeding, and understanding patterns of genetic diversity [1–4].
Haplotyping: Methods and Protocols by Irene Tiemann-Boege, Andrea Betancourt