RAPD in Microbial Molecular Identification

Methods used to distinguish bacterial strains include random amplified polymorphic DNA (RAPD) analysis, pulsed field gel electrophoresis, and ribotyping. RAPD is based on the amplification of genomic DNA with single primers of the arbitrary nucleotide sequences. Creative Biolabs has 10+ years of experience providing CRO services for live biotherapeutic product (LBP) development. Our skilled scientists can use RAPD technology to provide professional support for your microbial identification.

Principle of The RAPD Technique

The standard RAPD technique uses random sequences of short synthetic oligonucleotides (10 bases long) as primers to amplify total genomic DNA with nanogram amounts by PCR under low-temperature annealing conditions. The amplified products are usually separated by agarose gel and stained with ethidium bromide. At appropriate annealing temperatures during thermal cycling, random sequences of oligonucleotide primers bind several promoter sites to the complementary DNA sequences of the template genome. If the distances between these promoter sites are within the range of magnification, discrete DNA products will be produced. Nucleotide differences between different template DNA groups can lead to the presence or absence of bands due to the change of promoter sites. On average, each primer amplifies several discrete loci in the genome, so median loci are indistinguishable in RAPD models.

The principle of the RAPD technique.Fig.1 The principle of the RAPD technique. (Silva, 2004)

Applications of RAPD

  • Determination of Taxonomic Identity

By using different oligonucleotide primers, molecular signatures of diagnostic significance at different levels of classification can be generated. For instance, consider an RAPD analysis of several individuals within a species, and several species within a given genus.

  • Analyses of Interspecific Gene Flow and Hybrid Speciation

At the level of the individual, RAPD markers may be applied to parentage analysis; at the population or species level, RAPD may be used to detect hybrid populations or species. The detection of genome hybrids relies upon the identification of diagnostic RAPD markers for the parental genotypes under investigation.

  • Determination of Paternity and Kinship Relationships

Using polymorphic fragments between individuals, RAPD analysis can be used to assess parent-child relationships and relatability in large samples of offspring.

  • Analysing Mixed Genome Samples

RAPD techniques can be used to generate quantitative estimates of the relative proportions of different genomes in mixed DNA samples.

  • Generating Novel Specific Probes

Any diagnostic RAFD marker can be eluted from the gel, reamplified, radiolabelled with 32P, and serve as an inexhaustible supply of probe in Southern analyses.

Advantages and Disadvantages

RAPD is an easy and quick assay that requires low quantities of DNA (5-50 ng per reaction). The design of primers does not require sequence data because primers are randomly distributed throughout the genome. Each reaction produces multiple frequency bands, which are usually sufficient to reveal change. RAPD mode reading can be automated. The disadvantages of this technique are that a purified high molecular weight DNA is required. Due to the sensitivity of reaction conditions, highly standardized experimental procedures are required.

Creative Biolabs is a leading provider of custom LBP development services. Our team applies decades to offer tailored, flexible solutions matched to your needs. If you are interested in our RAPD technology, please contact us for more.

Reference

  1. Silva, G.J.F. Caracterização molecular de Bursaphelenchus xylophilus através de RAPD-PCR. 2004.

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