Using the plant DNA to be studied as a template, PCR amplification reaction is performed using synthetic primers.
High concentration agarose gel, non-denaturing or denaturing polyacrylamide gel electrophoresis to detect its polymorphism
How do I convert a fragment generated by a RAPD or SSR analysis into a SCAR (sequence-specific amplified region) marker?
(1) After RAPD analysis of genomic DNA, the target RAPD fragment is cloned and its end sequenced
(2) Design specific primers (18-24 bp) based on the sequences at both ends of the RAPD fragment. Generally, the first 10 bases of the primer should include the primers used for the original RAPD amplification.
(3) PCR specific amplification of genomic DNA using synthetic primers
9. How are enzyme-digested amplified polymorphic sequences (CAPS) generated? What is the basic procedure for performing a CAPS analysis?
The PCR-amplified DNA fragments were cut with restriction endonucleases and the cut fragments were separated by gel electrophoresis to obtain restriction endonucleases.
Sex fragment length polymorphism
Procedure: (1) PCR amplification using specific primers
(2) Digest the PCR amplification product with restriction endonucleases
(3) Separate the DNA fragments of the enzyme-digested products by agarose gel electrophoresis
(4) Ethidium bromide (EB) staining to observe fragment length polymorphism
10. What is a single nucleotide polymorphism (SNP)? What are the sources of SNPs? What are the characteristics of SNPs compared to other molecular markers?
Concept: refers to the variation of single nucleotides between different individuals' genomic DNA sequences. It is also known as the third generation of new polymorphic markers.
Sources of SNPs: single-base transitions, transversions, and single-base insertions and deletions
Features: (1) Bi-allelic markers: Single base substitutions include: 1 transition: CT (GA) and 3 transversions CA (GT), CG (GC), AT (TA) (the complementary chain is in brackets, and the left and right of the slash represent homologous chromosomes)
(2) Uneven distribution in DNA molecules: CT conversion occurs mostly at CpG methylation sites
(3) High density and extremely rich polymorphism. In the human genome, there is one SNP every 1.3kb.
(4) Genetic stability Based on single nucleotide mutation, the mutation rate is 10-9, which has higher genetic stability compared with SSR polymorphism.
(5) The detection and analysis of SNPs can be easily automated. For example, the application of DNA chip technology can achieve large-scale, automated analysis and detection.
11. Briefly describe the basis for constructing a genetic map.
During meiosis, homologous chromosomes undergo exchange, which results in the recombination of genes on the chromosomes. The frequency of recombination between two genes depends on the relative distance between them. Therefore, as long as the exchange value is accurately estimated and the relative position of genes on the chromosome is determined, a linkage map can be drawn.
What are the main steps in constructing a molecular genetic map?
(1) Select molecular markers suitable for mapping and determine parental combinations based on polymorphisms between genetic materials
(2) Establish a mapping population, that is, a segregating population or derivative line with a large number of molecular markers in a segregating state
(3) Analysis of marker genotypes of different plants or lines in a population
(4) Use computer programs to establish linkage relationships between markers
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