The homozygous genotype and heterozygous genotype are displayed to provide complete genetic information.
What categories can DNA molecular markers be divided into?
(1) Molecular markers based on DNA-DNA hybridization
(2) Molecular markers based on PCR technology
(3) Molecular markers based on a combination of PCR and restriction enzyme digestion
(4) Molecular markers based on DNA chip hybridization technology
(5) Molecular markers targeting specific domains
(6) High-throughput molecular labeling
3. Describe the principles and characteristics of restriction-length polymorphism (RFLP) markers and the basic steps for developing RFLP markers.
Principle: (1) Use specific restriction endonucleases to digest the genomic DNA of different organisms. The number of DNA fragments produced and the length of each fragment reflect the distribution of different restriction endonucleases on the DNA molecule.
(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 products are randomly amplified. Due to the use of shorter random single primers and low annealing temperature, on the one hand, the stable pairing of nucleotide primers and templates is guaranteed, and on the other hand, appropriate mismatches are allowed, thereby expanding the range of primer pairing in genomic DNA and improving the efficiency of genomic DNA analysis.
5. What is amplified fragment length polymorphism (AFLP)? How does the selective restriction fragment amplification produced in the AFLP reaction work?
AFLP marker: It is a molecular marker technology that combines restriction endonuclease digestion of DNA with PCR technology to produce fragments of different lengths to detect DNA polymorphisms.
principle:
Why is it necessary to perform two consecutive PCR amplifications in AFLP analysis?
The two-step amplification reaction makes the amplification results clearer and more reproducible
The basic principles of SSR markers and the general procedures for establishing SSR markers.
Basic principle: Since the flanking sequences of a specific microsatellite in the genome are usually single sequences that are highly conserved, the DNA fragments flanking the microsatellite can be cloned and sequenced. Then, primers are artificially synthesized based on the complementary sequences at both ends of the microsatellite's flanks, and the microsatellite fragments are amplified through PCR. Due to the different number of tandem repeats of the core sequence, products of different lengths can be amplified by PCR. The amplified products are subjected to electrophoresis, and the variation in the length of the amplified products of different individuals produces length polymorphism, which is also called simple sequence repeat length polymorphism (SSLP).
Procedure: (1) Establishment of genomic DNA library
Design and synthesize oligonucleotide probes based on the obtained SSR types, and screen the desired recombinant clones by colony hybridization
Sequencing of DNA insert sequences of positive clones
Design and synthesize primers based on the sequences on both sides of the SSR
This chapter is not over yet, please click on the next page to continue reading!
Continue read on readnovelmtl.com
