When we examine their second copies of this RFLP, we see that they are not identical. Jack 2 lacks an EcoR I restriction site that Jill has 1.2 kb upstream of the target sequence (difference in italics).
The male can only produce one type of gamete (1 and 2) but the female can produce four different gametes. Two of the possible four are called parental because they carry both RFLP bands from the same chromosome; 1 and 2 from the left chromosome or 3 and 4 from the right chromosome. The other two chromosomes are recombinant because recombination has occurred between the two loci and thus the RFLP bands are mixed so that 1 is now linked to 4 and 3 is linked to 2.
Therefore, when Jack and Jill have their DNA subject to RFLP analysis, they will have one band in common and one band that does not match the others in molecular weight:
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These loci are located on the same chromosome for the female (left) and the male (right). The upper locus can produce two different bands called 1 and 3. The lower locus can produce bands called 2 or 4. The male is homozygous for band 1 at the upper locus and 2 for the lower locus. The female is heterozygous at both loci. Thier RFLP banding patterns can be seen on the Southern blot below:
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8.2 Choice of restriction enzymes to use for RFLP detection
With so many restriction enzymes available, how does one decide which ones are the best to use in the search for RFLPs? Obviously, cost is an important consideration. Another consideration is whether the enzyme is optimally active with genomic DNA obtained from animal tissues. 57 However, a critical consideration is the rate at which RFLPs can be detected based on the enzyme that is chosen.
A systematic study of RFLP detection between B6 and M. spretus DNA subsequent to digestion with one of ten different enzymes has been reported by LeRoy et al. (1992). One hundred and ten anonymous DNA sequences of less than 4 kb in length were used as probes. The highest rate of RFLP detection 63% was observed with DNA digested with TaqI. The second highest rate 56% was observed with MspI. In decreasing order of effectiveness were the enzymes BamHI (50%), XbaI (47%), PstI (44%), BglII (41%), Hind III (39%), PvuII (38%) Rsa I (38%), and Eco RI (33%). It is ironic that of the ten enzymes tested, the one most commonly used in molecular biological research EcoRI was the worst one, by a long shot, at detecting polymorphisms.
A theoretical explanation for the observation that TaqI and MspI are more likely than other enzymes to detect RFLPs can be found in the dinucleotide CpG which is at the center of both recognition sites. This dinucleotide is unusual in two respects. First, it is present in mammalian genomes at a frequency one-fifth of that expected from base composition alone. Second, when it is present, the cytosine within the dinucleotide is usually methylated. 58 As it turns out, the latter fact explains the former because methylated cytosine has a propensity to undergo spontaneous deamination to form thymidine. This complete transition is not recognized as abnormal by the repair machinery present in mammalian cells, and thus methylated-CpG dinucleotides serve as one-way hotspots for mutation (Barker et al., 1984). As a consequence, the CpG dinucleotide is relatively rare, and when it does occur in a methylated form, it is more likely to mutate than any other dinucleotide. Even an unmethylated CpG can undergo a spontaneous mutation from cytosine to uracil; however, this abnormal nucleotide is more likely to be recognized and repaired. Nevertheless, in those few cases where repair does not occur, the uracil will basepair with an adenosine in the following round of DNA replication, leading to the same substitution as found with methylated CpGs.
Thus, TaqI and MspI are the most useful enzymes for the identification of RFLPs. Both enzymes recognize four basepair sites, TaqI recognizes TCGA and MspI recognizes CCGG. If nucleotides were randomly distributed across the genome, TaqI and MspI sites would be distributed at average distances of 270 bp and 514 bp, respectively. 59 However, as a consequence of the paucity of CpG dinucleotides, these two restriction enzyme sites are actually found much less frequently in mammalian DNA. Empirical data indicate restriction fragment size distributions that average 2.9 and 3.5 kb for TaqI and MspI respectively (Barker et al., 1984).
In practice, the enzyme TaqI is the better choice of the two for use in RFLP analysis. It is relatively cheap and it works well with animal DNA samples that other enzymes refuse to cut (presumably aided by the high temperature at which the digestion is carried out). MspI is somewhat more sensitive to contaminants within animal tissue DNA samples, but is a good second choice. When the results obtained with TaqI and MspI are combined, the Gu´net group detected RFLPs at 74% of the loci tested for variation between spretus and musculus (LeRoy et al., 1992). When the results obtained with XbaI were added in, 79% of the loci were polymorphic. When the results obtained with the remaining seven enzymes were included, RFLPs were detected at 83% of the loci. The take-home lesson from this study is that it is most cost-effective to search for RFLPs on standard 1% agarose gels with just three enzymes TaqI, MspI, and XbaI. If the search is unsuccessful at this point, it would appear that the locus under analysis is not highly polymorphic at the DNA level, and in those cases where the locus is just “one more marker,” it is probably not worth pursuing further. On the other hand, if the locus is of importance in and of itself, it makes sense to pursue more sensitive, PCR-based avenues of polymorphism detection such as ingle-strand conformation polymorphism (Section 8.3.3) or linked microsatellites (Section 8.3.6).
FAQ
How is RFLP used to identify a person?
Why do we use RFLP?
What does the P stand for in RFLPs?
How are RFLP made?
The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from 500 to 2000 bp are excised, eluted and cloned into a plasmid vector (for example, pUC18). Digests of the plasmids are screened to check for inserts.
