Polymerase Chain Reaction-Based Markers

Abstract

Restriction fragment length polymorphism (RFLP) was the first DNA-based marker, and it was once widely used in biology and, to some extent, plant breeding. But due to the need for high technical skill, considerable marker development work, and some other limitations, the search continued for more user-friendly DNA marker systems. With the discovery of polymerase chain reaction (PCR) technique, the new generation of PCR-based DNA markers was developed. Initially arbitrary primers of different sizes were used to amplify genomic DNA to generate fingerprints of different individuals. Randomly amplified polymorphic DNAs (RAPDs), DNA amplification fingerprinting (DAF), and arbitrary-primed PCR (AP-PCR) are examples of marker systems based on arbitrary primers. Amplified fragment length polymorphism (AFLP) marker system detects polymorphism due to the sequence variation in and around the recognition sites of restriction endonucleases and uses PCR for marker assay. Refinements in the DNA sequencing technology supported the discovery and development of marker systems, which exploit the sequence variation in specific fragments of DNA using the PCR technology. Sequence-tagged site (STS) markers, including microsatellite or simple sequence repeats (SSR) markers, are an example of this group. The SSR markers revolutionized the marker application in crop improvement in view of their abundance, codominant nature, user-friendliness, and other desirable features. But the development of SSR markers is expensive and complicated so that several other simpler PCR-based marker systems like sequence-related amplification polymorphism (SRAP; it uses open reading frame sequences), target region amplification polymorphism (TRAP; it uses expressed sequence tags), etc. were developed. In addition, some markers exploit variation in RNA sequences of a species: cDNA AFLP, cDNA-SSCP, etc. are examples of RNA-based markers. This chapter describes these and other PCR-based marker systems in some details in addition to introducing the technique of PCR.

Appendices

Appendices

1.1 Appendix 3.1: The Number of RAPD Bands Theoretically Expected from a DNA Sample

The number of RAPD bands theoretically expected from a DNA sample can be estimated on the basis of probability concept. It can be shown that the number of RAPD bands (b) of a given average size (f bp) expected from a genome of known size (N bp) amplified using primers of n nt would be given by the following formula:

$$ b=2Nf/{16}^n $$

(3.1)

The above formula is derived as follows. The probability that a specified base would occur at a given site in a DNA strand will be 1/4 since this site could have any one of the four DNA bases. It is assumed that the distribution of nucleotides/bases is random, i.e., governed by chance, so that the four DNA bases occur in the DNA molecule in equal proportion. Surely, this assumption is unrealistic, but it is necessary for an easy estimation of the above and similar parameters. Therefore, the probability that the n bases present in a RAPD primer will be found in a DNA strand will be 1/4ⁿ. Exponential amplification can occur only when a second primer binding site occurs in the neighborhood of the first site; the probability of the two primer binding sites occurring together will be 1/4²ⁿ or 1/16ⁿ. Since the template DNA has two complementary strands, the primer binding sites could occur on either strand at a given site. In addition, the two primer binding sites would be separated by f bp, i.e., the RAPD fragment size. Therefore, the probability of two primer binding sites occurring in a DNA duplex of f bp would be 2f/16ⁿ. If the size of genomic DNA were N bp, the number of expected RAPD fragments of f bp would be 2Nf/16ⁿ.

According to the above formula, a primer of 10 bases is expected to generate 2 bands in rice, which has the genome size of 450 Mb and 4 bands in tomato that has genome of 950 Mb. Similarly, it would produce 9 bands in maize (genome size, 2,500 Mb) and 19 bands in barley (genome size, 5,300 Mb).

1.2 Appendix 3.2: Polymerase Chain Reaction and Randomly Amplified Polymorphic DNAs

PCR was developed for amplification of a specific segment from a DNA sample of high complexity, e.g., human genomic DNA. Subsequently, this procedure was applied to achieve a variety of other objectives, for each of which the procedure was suitably modified. In a general sense, the term PCR signifies repeated replication of a segment of sample DNA by using suitable primer(s) and DNA polymerase. In this sense, all applications of the technique would qualify as PCR. But in a restricted sense, the term PCR signifies amplification of a specific sequence from the sample DNA; this PCR procedure differs in many ways from the other applications of the technique. The various features of PCR (in the restricted sense) and RAPDs are summarized in Table 3.3.

Table 3.3 A comparison between PCR and RAPD procedures