RAPD (Random Amplified Polymorphic DNA) analysis technique

Summary

Polymerase chain reaction (PCR) has greatly influenced almost all areas of molecular biology with its superiority and with its basic procedure and DGGE, a variety of methods have been developed to detect nucleotide variation.RAPD (Random Amplifed Polymorphic DNA) is one of them, which can be used for (1) construction of genetic maps (2) systematic studies (3) Marking and localization of target genes (4) Detection of purity and exogenous genes.

Operation method

RAPD analysis techniques

Principle

RAPD as a single-primer PCR amplification product, its mechanism is that the primer first binds to the template strand, along the template 5'-end direction to produce different lengths of DNA fragments, and then use these fragments as a template to continue to a large number of amplification. Because of the RAPD reaction, there is no corresponding primer and template complementary at the 3'end, so there must be a primer along the template DNA 5'-end direction of the extension of the 3'end of the formation of the same primer complementary sequence of the process. It has been proposed that in the process of RAPD reaction, there may be some molecular modes of amplification: (1) The single-stranded DNA molecule with primer at the 5' end is folded, binds and extends itself at the 3' end and forms the complementary sequence of the same primer at the 3' end, which becomes the template molecule for single-primer PCR amplification after denaturation and unfolding. (2) It can be realized by the colligation of two single-stranded DNA molecules each with the same primer at the 5' end. (3) For the inverted repetitive sequences of genomic DNA molecules, if the binding position of the primer is exactly at the 3' end of these sequences, then each of the two single-stranded DNA fragments has a binding position of the primer and its complementary sequences, which become the template molecules for RAPD amplification. This is rarely the case in two-primer universal PCR, but because the primers used for RAPD are short and annealed at low temperatures, this increases the chances that the primers will bind to the inverted repeat sequences, and it is entirely possible that a RAPD will result.

Materials and Instruments

Template DNA
Oligonucleotide primers dNTPs Taq DNA polymerase PCR buffer Mineral oil Reagents for electrophoresis
PCR amplifiers Electrophoresis units Microcentrifuges Micro pipettes Eppendorf tubes UV viewers and photographic equipment

Move

I. Test conditions
1. Drugs and reagents
(1) Template DNA (10-100 ng)
(2) Oligonucleotide primers (20 mmol/L storage solution, available from Operon Technologies or Perkin Elmer, or synthesize your own)
(3) 0.2 mmol/L dNTPs (It is important to use high quality dNTPs. dNTPs degrade upon repeated freezing and should therefore be stored in small portions. Care should be taken that the amounts of the four dNTPs in the mixture are equal)
(4) Taq DNA polymerase (Perkin Elmer, Norwalk, Connecticut)
(5) 10xPCR buffer (500 mmol/L KCl, 15 mmol/L MgCl₂, 100 mmol/L Tris HCl, pH 8.3)
(6) mineral oil
(7) Reagents required for electrophoresis
2. Instrumentation
PCR amplifier, electrophoresis device, microcentrifuge, micropipettes (1-20 ml and 20-200 ml), 0.5 ml Eppendorf tubes, UV observation device and photographic equipment.
II. Operating Procedures
1. Add the following reactants to a sterile Eppendorf tube in ice:Water: 14.75 ml10x buffer: 2 ml10xdNTPs: 2 mlPrimer (20 mmol/L): 0.2 mlTaq DNA polymeraseFinal VolumeDNA (10-100ng): 1 mlParaffin oil : 20 ml
2. Start the following cycle after reaction at 93°C for 2 min:Denaturation reaction at 93℃: 1 minAnnealing reaction at 36℃: 1 minExtension reaction at 72℃: 1.5 minAfter 45 cycles, the last cycle of 72°C was increased for 5 min, and the reaction products were stored at 4°C at the end of the cycle.
3. 20 ml of reaction product was subjected to gel electrophoresis and the amplification was detected by ethidium bromide staining.

Caveat

I. Problems to be noted and solutions1. Amplification deviation or no amplification.(1) A component is missing in some or all tubes. Repeat a small number of several reactions to determine if all PCR components have been added.(2) PCR inhibitors may have been purified with the DNA, altering the concentration of DNA.(3) Include a wash step (e.g., phenol extraction) in the DNA isolation.(4) Dilute the new DNA solution.(5) Failure of the primer master mix. Reconstitute the master mix, use a different primer or increase the primer concentration.(6) Extend the annealing time (in the second part of the PCR program), or decrease the rate between warming conversion steps.(7) Increase the concentration of Taq polymerase in each reaction.(8) Make up new components and autoclave any that should be sterilized.

2. Poor amplification results with blurred or illegible bands.(1) Replace the Taq polymerase buffer.(2) Check the primer (use another primer, or label the end of the primer and detect it by electrophoresis on 16% 6 mol/L urea polyacrylamide gel).(3) Check Taq polymerase activity (compare with different batches of enzyme).(4) Change the DNA concentration.

3. High relative molecular mass product diffusely distributed >4 kb.(1) Decrease DNA concentration.(2) Reduce Taq polymerase concentration.(3) Decrease the amount of gel sampling.(4) Possibly as a result of an excessive number of cycles (Bell and Demarini 1991), reduce the number of cycles.(5) Confirm that the correct buffer is being used (not water!) to prepare the gel.(6) Electrophoresis at a lower voltage.

4. A single, strong band is present in the control and all samples tested.Confirm that the primer sequence is not a palindrome. This may be the result of primer multimerization. Use a different primer.

5. The band profile is not reproducible.Too much or too little DNA. Limit the concentration of genomic DNA to 10-100 ng. Too little DNA results in inefficient binding of the "true" target sequence to the primer, so primer amplification produces false bands, called primer artifacts; too much DNA results in mispairing (pairing of the primer with the genomic DNA). Two reactions with different DNA concentrations were performed for each sample. Only the major bands are considered.

6. Too much gel background after staining affects resolution.(1) Reduce the staining time.(2) Extend the gel destaining time.(3) Too much DNA or Taq polymerase in the PCR reaction.

7. Insufficient separation of low relative molecular mass products.Separate products on a higher concentration agarose gel, specialty pure gel, or polyacrylamide gel.

Common Problems

1. RAPD has the following characteristics: ① There is no need to specially design primers for RAPD amplification reaction, and there is no need to predict the nucleotide order of the genome of the organism under study, the primers are randomly synthesized or arbitrarily selected. The primers are randomly synthesized or arbitrarily selected. The length of primers is generally 9-10 oligonucleotides. ② In each RAPD reaction, only a single primer is added, and amplification is realized by random pairing of primers and template DNA strands, and amplification has no specificity. (iii) The annealing temperature is low, generally 36°C, which ensures stable pairing of short nucleotide primers with the template and also allows for appropriate mispairing to expand the randomness of primer pairing in genomic DNA. ④ Compared to conventional PCR, RAPD reactions are easy to program. Using a set of random primers, a large number of DNA molecular markers are obtained, which can be systematically analyzed with the help of a computer.

2. The advantages and shortcomings of RAPD analysis, RAPD is a new and effective genetic markers, compared with other molecular markers, has many advantages: ① Synthesize a set of primers, can be used for the analysis of different biological genomes. Comparatively speaking, RFLP markers are race-specific, which limits its application. ② RAPD technology is simple and easy to implement, saving labor and time. It does not need the preparatory work for RFLP analysis, such as clone preparation, isotope labeling, Southern blotting and molecular hybridization. Moreover, RAPD detection is sensitive and convenient, with fluorescent dyes or isotope labeling can be detected, which greatly increases the speed of its analysis. Even if we use sequence gel to analyze RAPD, one person alone can complete the analysis of 120 samples in only 36 hours, while RFLP takes at least one week. (iii) The amount of sample DNA required for RAPD analysis is extremely small. It is only 1/1000-1/200 of RFLP, which is beneficial for early sampling and identification of organisms or when DNA is restricted. ④ Since RAPD does not require cloning when used to construct gene maps, this can break the limitations of cloning vectors and hosts, expanding the scope of application. ⑤ Each RAPD marker is equivalent to a target sequence site in genome analysis, which can simplify the process of transferring information for collaborative research projects. (6) RAPD markers can make genetic linkage maps of genomic regions that are difficult to distinguish by RFLP. (7) RAPD analysis can be automated, which reduces the troublesome procedures like RFLP.

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