What is rflp how are they made

The digested DNA is size-fractionated on a preparative agarose gel, and fragments ranging from to bp are excised, eluted and cloned into a plasmid vector for example, pUC Digests of the plasmids are screened to check for inserts. Southern blots of the inserts can be probed with total sheared DNA to select clones that hybridize to single- and low-copy sequences.

You are here: NCBI. External link. Upon resolving on an agarose gel, genomic DNA that does not hybridize with the probe will obscure the locus of interest as a large smear. A filter is placed on top of the agarose and pressed against it to transfer the DNA in a process called Southern Blotting.

Following a lengthy transfer, the filter is denatured to and incubated with the radioactive probe. To visualize this probe hybridization, a film is exposed to the filter and processed. Following restriction digestion, the samples are resolved on an agarose gel. Digestion of genomic DNA will result in a large smear. Following transfer of the DNA onto a membrane through capillary action, the membrane is probed with radioactive probe DNA. The probe binds selectively to complementary sequences to reveal a series of distinct bands.

An interactive demonstration of the first DNA fingerprinting. Sample A only reveals one band after processing because this person is homologous for the same allele. Sample B is heterozygous and reveals three bands. RFLPs represent inheritable markers and can reveal relationships between different individuals. A pedigree can illustrate the relationship of the inherited alleles. The technique can be more informative if using multiple probes simultaneously for different loci or to use multi-locus probes that hybridize to multiple locations.

While RFLPs can arise from SNPs, they may also be caused by the expansion or contraction of repeated elements between restriction sites. What is the role of the dye in these samples? Should we be alarmed that the samples are all the same color? What does it mean that there are multiple bands in a lane? I ; Anete P. The past limitations associated with pedigree data and morphological, physiological and cytological markers for assessing genetic diversity in cultivated and wild plant species have largely been circumvented by the development of DNA markers such as restriction fragment length polymorphisms RFLPs; Botstein et al.

However, these molecular markers have technical differences in terms of cost, speed, amount of DNA needed, technical labor, degrees of polymorphism, precision of genetic distance estimates and the statistical power of tests. Although the discrimination power of RFLPs in diversity studies has been well documented Smith et al. Microsatellites SSRs occur frequently in most eukaryote genomes and can be very informative, multiallelic and reproducible Vos et al.

The application of SSR techniques to plants depends on the availability of suitable microsatellite markers, which have been developed for species such as soybean Rongwen et al. Morgante and Olivieri stated that in soybean the amount of information given by SSR loci in relation to a comparable number of RFLP loci is given by the estimated number of alleles 4. The AFLP technique is more laborious and time consuming than RAPD methods but is also more reliable, AFLP being able to detect a large number of polymorphic bands in a single lane rather than high levels of polymorphism at each locus such as is the case for SSR methods.

Although this lower sensibility in detecting informative genotypic classes might be associated with the inability to distinguish heterozygotes from homozygotes because of binary scored AFLPs, Gerber et al. Comparisons of different DNA markers for diversity studies in maize Hahn et al. However, in the case of maize, tropical and temperate populations differ from each other because tropical populations usually originate from composites with higher genetic variability and, most of the time, it is difficult to allocate tropical composites to well-defined heterotic groups by phenotypic evaluation.

Due to this uniqueness, molecular markers have been very useful in genetic evaluations and assignment of tropical maize inbred lines to heterotic groups. The objectives of the study described in this paper was: i compare the level of information provided by RFLP, RAPD, SSR and AFLP markers for estimating genetic similarities in tropical maize inbred lines; ii evaluate the minimum number of loci of each marker needed to accurately represent genetic distance between inbred lines; iii compare the genetic distances GD obtained with the different marker system; iv compare the usefulness of these four markers in predicting single-cross hybrid performance by means of genetic distance estimates.

Eighteen S 3 selected inbred lines from two divergent tropical maize populations eight from BR and ten from BR previously had their genetic distances surveyed using four different marker systems Lanza et al. Detailed descriptions of these populations are given in Lanza et al.

Total genomic DNA was isolated from a bulk of five-week-old leaf tissue taken from 16 plants of each line, then being isolated and purified by the method of Hoisington et al.

Thirty-two primers showing reproducible polymorphism were selected and used for scoring the 18 inbred lines. When performing RAPD analysis, each band was considered as one locus. Briefly, a total of clone-enzyme combinations were analyzed, the maize genome being saturated 20 cM intervals with at least one RFLP probe selected by its map location on each chromosome. Barbosa et al. For the AFLP method 20 primer combinations were used and binary scored 1 or 0 with each band being considered a locus while for the SSR method 68 polymorphic primers were used with the binary data being converted into a genotypic matrix which was used to identify alleles and their respective loci.

Both dominant markers RAPD and AFLP were used to calculate the genetic distances between the 18 inbred lines using the complement of the Jaccard's similarity coefficient Jaccard, which takes into account the presence or absence of bands. In this method, co-occurrences are divided by the total number of evaluated loci excluding the negative co-occurrences and thus can be interpreted as the proportion of coincidences in relation to the total number of evaluated loci.

Jaccard similarities were calculated using version 2. The genetic distances for the codominant markers RFLP and SSR were calculated using the modified Roger's distance MRD; Goodman and Stuber, based on the allele frequency of each locus which considers the amount of genetic diversity and expresses the quantity of diversity present in each locus or allele, calculations being made using version 1. Pearson's correlation coefficient was calculated for the genetic distances, single cross performance and heterosis as previously described by Benchimol et al.

The information content of each marker system was calculated for each marker and locus using the polymorphism information content PIC Lynch and Walsh, which provides an estimate of the discriminating power of a locus by taking into account not only the number of alleles that are expressed but also their relative frequencies.

Calculations were made using the following formula: where f i is the frequency of the i th allele. Bootstrap analysis was used to verify if the number of polymorphic loci evaluated was high enough to provide accurate genetic distance estimates King et al. To determine the sampling variance of the genetic distances produced by the different molecular data sets we performed bootstrap analysis using a decreasing number of loci for codominant markers or bands for dominant markers.

For each specific number of loci or bands used the polymorphic markers were submitted to random samplings with replacement bootstrap samples and genetic distances were obtained for each bootstrap sample Tivang et al.

Each band visualized on the gel was considered to be the re-sampling unit for dominant markers because for these markers each band is related to one locus. Codominant markers relate each band to an allele, and therefore the boostrap was applied among locus. The coefficient of variation CV for all genetic distances across the bootstrap samples was estimated for each specific number of loci or bands sampled, a computer program for performing these analyses being set up using the 'RANNUNI' function of the SAS system Version 8.

We used the median and maximum coefficient of variation values to evaluate the accuracy of the genetic distance estimates because although the mean coefficient of variation is often used in the literature caution is needed when dealing with molecular marker data for which there is no assurance that the CVs values are distributed symmetrically. All of the 18 maize inbred lines studied by us had previously been investigated using the four different marker systems RAPD: Lanza et al.

In the work of Lanza et al. Differences in the distribution profiles also occurred between dominant and codominant markers, with dominant markers having higher standard deviations than codominant markers.

The RAPD markers were clearly the most distinct type of marker because the correlation values involving this marker were equal to or lower than 0.

As expected, the magnitude of the coefficient of variation CV values decreased as the number of polymorphic loci bands evaluated increased. Within each sample i. Because the mean is not a good indicator of central tendency for skewed data we calculated the minimum number of loci necessary for an accurate representation of the genetic distances by fitting an exponential function based on the mean, median and maximum CV values of the genetic distances obtained by bootstrap sampling to the data for each marker, the results of this analysis being given in the Boxplots shown in Figure 3.

The results obtained based on the adjusted functions except for the mean CV shown in Figure 3 are presented in Table 3. Similar patterns were observed for both genetic distance and heterosis. Although similar average genetic distance values were obtained for the BR and BR intrapopulation crosses, the BR crosses showed the widest range of genetic distances with all of the four different markers assayed; probably because of the broader genetic base of the BR population.