One of the early polymerase chain reaction (PCR) based DNA markers develops was RAPD (Randomly Amplified Polymorphic DNA) marker. In a Q & A session of the AGH635 lecture, one of the student asked the following question: “What is RAPD marker and how can one generate the marker?”
Before posting the answer to that question, let’s remind you what the DNA is. As everyone already knows, the DNA is simply a polymer of a nucleotide (a molecule consisted of phosphate-sugar-nitrogen base). The nitrogen base portion consisted of four different types, such as: Adenine (A), Cytosine (C), Guanine (G) and Thymine (T). If we look at the nitrogen part, the primary structure of DNA is actually a stretch of A, C, G, T sequences in particular orders and certain direction.
If we consider that a DNA consisted a random nucleotide sequences (however, it is not random at all in nature!), then the probability of getting a stretch of 10 bases consisted of A, C, G, and T in a random order (such as: “ACAGCTAGAG”) would be approximately (0.25)*(0.25)*(0.25)*(0.25)*(0.25)*(0.25)*(0.25)*(0.25)*(0.25)*(0.25)=9.53674*E-07. Therefore, any random 10 base sequences would be found in a frequency of once in every 95,367,400 bases (once in 95 million bases; Figure 1.A).
However, since the sequences of the genomic DNA is not completely random, it should be possible to find a 10 bases of a random nucleotide sequence such as “ACAGCTAGAG” in more frequent manners in the genome (Figure 1.B).
Those cases in Figure 1 are the bases of the RAPD markers development. Moreover, if ones explore further about the random sequences and the genomic DNA, one would find the following :
- A random – 10 base sequences can be found interspersed in different part of the plant genome and randomly distributed throughout the genome as illustrated in Figure 2.
- The random sequences can be in the same orientation (i.e. reside in the same strand, such as position 1, 3, 5, and 7) or in the opposite direction (i.e. reside in different strands, such as position 1 & 2; 3 & 4; 3 & 6; 4 & 6; and 5 & 6) as illustrated in Figure 3.
- The positions of two random sequences can be separated by a few hundreds or thousands bases apart as illustrated in Figure 3.
- Two random sequences, that are in opposite direction and flanking DNA sequences of less than 3,000 bases, can be amplified using a single random oligonucleotide primer and generate a certain size of PCR product. The PCR product can be used as DNA marker, also known as randomly amplified DNA marker.
- The DNA marker most of the time is polymorphic among different individuals, as such it is called randomly amplified polymorphic DNA (RAPD) marker.
Finally, the random sequences, such as position 5 & 6, as illustrated in Figure 4, are usually present in a number of locations in the genome.Therefore, if a single random oligonucleotide (i.e. ‘acagctagag’) is used to PCR amplify target DNA using plant genomic DNA as template, then it should be possible to amplify a number of DNA fragments. Therefore, in one PCR reaction – a number of RAPD markers can be generated using a single random oligonucleotide primer. An example of RAPD markers generated using a single random oligonucleotide is presented in Figure 5.
In the above example (Figure 5), a single OPA9 primer are used to amplify RAPD markers in Calamus palustris. Among samples of C. palustris analyzed in the above figure, the primer OPA9 generates either one, two or three DNA fragments. As such, in the Figure 5, a single OPA9 primer generates 1 – 3 RAPD markers.