(2) Agarose gel electrophoresis separates these fragments in order of size, and then transfers them to easy-to-operate nylon membranes and nitrocellulose membranes in their original order and position.
(3) Use DNA labeled with radioactive nuclides (such as 32P) or non-radioactive substances (such as biotin, digoxin, etc.) as a probe to hybridize with the DNA on the membrane
(4) Radioautography or enzymatic detection can show the polymorphism of restriction enzyme fragments of the probe in different materials
Characteristics: Basic characteristics: (1) The detected alleles have codominant characteristics
(2) The number of RFLP marker sites is not limited, and the number of gene loci that can be detected is usually 1 to 4.
(3) RFLP probes mainly come from three sources: cDNA clones, plant genome clones (RG clones), and PCR clones.
Advantages of RFLP: (1) Co-dominant markers can identify heterozygous and homozygous genotypes
No sequence information of the detection object is required
Disadvantages: (1) The probes used in RFLP analysis must be single copy or oligo copy, otherwise they cannot show clear and identifiable band patterns.
(2) The amount of sample DNA required for detection is large (5-15 μg)
(3) The experimental operation is relatively complicated, and the cost is high when testing a few probes
(4) If radioactive nuclides (usually 32P) are used in the test, it is easy to cause environmental pollution and the test cycle is long.
(5) The hybridization signal of non-radioactive labeling systems (such as Biotin system, Dig system and ECL system) is relatively weak, the sensitivity is low, and the price is relatively high.
Basic steps: (1) DNA extraction
(2) Restriction endonucleases cut DNA
(3) Separation of DNA fragments by gel electrophoresis
(4) Transfer DNA fragments to the filter membrane
(5) Using radioactive (or non-radioactive such as chemiluminescent) labeled probes
(6) Southern hybridization
(7) Autoradiography reveals specific DNA fragments
(8) Analysis results
4. What is the principle behind the generation of random amplified polymorphisms (RAPDs)? Compared to conventional PCR marker reactions, what are the characteristics of the PCR reaction system used to generate RAPDs?
Principle: Using random oligodeoxynucleotide single-stranded fragments (usually 8-10 bp in length) as primers (also called random primers), PCR is used to amplify the DNA in the chromosome group to obtain DNA fragments of different lengths. The amplified fragments are then separated by gel electrophoresis and stained to show the polymorphism (length difference) of the amplified fragments.
Amplified fragment polymorphism reflects the DNA polymorphism of the corresponding region of the genome
Features: (1) Primer: single, 8-10 bp in length
(2) Reaction conditions: The annealing temperature of classic PCR is relatively high, generally 55-60°C, while the annealing temperature of RAPD is only around 36°C.
(3) Amplification product: RAPD product is random amplification. Due to the use of short random single primers and low annealing temperature, a
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