Why is rflp so accurate

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Genotyping Developing RFLP probes Total DNA is digested with a methylation-sensitive enzyme for example, PstI , thereby enriching the library for single- or low-copy expressed sequences PstI clones are based on the suggestion that expressed genes are not methylated. In this review, we mainly focus on the introduction of several important DNA-based markers, and their various applications in characterizing animal genetic resources.

RFLP is a method established by Grodzicker et al. Its basic principle is as follows: first, genomic DNA from different individuals is digested into DNA fragments of varying size, using known restriction enzymes.

Second, the digested fragments are separated via electrophoretic analysis. Finally, separated fragments are hybridized with radioactive or chemiluminescent homologous probes and exposed to an X-ray film; the different fragments are visible by autoradiography.

The molecular basis of RFLP is that nucleotide base substitutions, insertions, deletions, duplications, and inversions within the whole genome can remove or create new restriction sites.

RFLP was the first DNA-based marker for constructing genetic linkage maps; it is also one of the most widely used markers in AnGR assessments and breeding program development. The main advantages of RFLPs include: 1 high reliability, because it is generated from specific sites via known restriction enzymes and the results are constant over time and location. The disadvantages of RFLPs are as follows: 1 labor-intensive and time-consuming.

RAPD was developed by U. It amplifies the target genomic DNA with short, arbitrary primers commonly 10 bp in a PCR reaction, and can be used to produce relatively complicated DNA profiles for detecting amplified fragment length polymorphisms between organisms.

Since the arbitrary primers complement different parts of the genomic DNA, PCR products will differ in number and size polymorphism. For example, the RAPD method was used to generate specific fingerprint patterns of ten different species: including wild boar, pig, horse, buffalo, beef, venison, dog, cat, rabbit, and kangaroo [ 16 ].

RAPD markers have several obvious features as summarized in the literature: 1 no prior sequence knowledge is necessary for designing the specific primers, which can then be used in different templates. The AFLP procedure is as follows: first, the genomic DNA is digested with a restriction enzyme, and then the digested fragments are ligated to synthetic adaptors and amplified with specified primers that are complementary to a selective sequence on the adaptors.

Subsequent separation of the amplified fragments is obtained by selective primers and visualized using autoradiography [ 21 ]. Hoda et al. They analyzed 93 unrelated individuals from three local Albanian sheep breeds markers. The results obtained indicated high diversity in Albania sheep breeds [ 22 ]. AFLPs are notable for their genetic stability, they provides an effective, rapid, and economical tool for detecting a large number of polymorphic genetic markers, that can be genotyped automatically [ 23 , 24 ].

However, AFLPs are dominant bi-allelic markers [ 23 ], and are unable to distinguish dominant homozygous from dominant heterozygous individuals [ 25 ]. The AFLP method is an ideal molecular approach for population genetics and genome typing, it is consequently widely applied to detect genetic polymorphisms, evaluate, and characterize animal genetic resources [ 26 — 29 ].

Generally they consist of motifs which are made up of 1—6 base pairs bp tandemly repeated several times e. The flanking regions of repeated sequences at microsatellite loci are mostly conservative and the repetition motifs are highly variable between different species and even different individuals of the same species.

So we can design specific primers based on the conserved sequences and amplify the core repeat sequences by way of PCR, genetic polymorphisms can then be detected via electrophoresis [ 31 ]. Until recently, microsatellites were the markers most widely used for genetic diversity, mapping quantitative trait loci for production, and functional traits in farm animals [ 32 — 34 ]; they have also been used for marker assisted selection practices [ 35 ].

The advantages and disadvantages of SSR markers have been reported by many authors [ 36 — 40 ]. Its advantages are as follows: low quantities of template DNA required 10— ng , high polymorphism, co-dominant markers, high accuracy, high reproducibility, different microsatellites can be multiplexed in PCR, and they are amenable to automation.

Its disadvantages include: time-consuming and expensive to develop, heterozygotes may be misclassified as homozygotes when null-alleles occur because of mutations in the primer annealing sites, stutter bands may complicate accurate scoring of polymorphisms, underlying mutation model largely unknown, and microsatellite markers do help to identify neutral biodiversity but do not provide information on functional trait biodiversity.

Despite these disadvantages, microsatellite markers are still popular nuclear DNA markers for the investigation of genetic variation among and within species. In addition to the classical markers discussed above, with the development of modern molecular techniques and the completion of the Human Genome Project HGP , some new markers have emerged and are being used in the evaluation of farm animal genetic resources; these include high-density SNP arrays, whole-genome sequencing, and DNA barcoding.

SNP, a novel molecular marker technology, was first proposed by Lander in , it refers to a sequence polymorphism caused by a single nucleotide mutation at a specific locus in the DNA sequence.

Of all the SNP mutation types, transitions are the most common approx. Currently, SNP markers are one of the preferred genotyping approaches, because they are abundant in the genome, genetically stable, and amenable to high-throughput automated analysis [ 42 ].

SNPs are bi-allelic markers, indicating a specific polymorphism in only two alleles of a population [ 44 ]. SNPs distribute in both coding and non-coding regions of genomes, they are vital players in the process of population genetic variations and species evolution [ 45 ]. SNPs are third generation molecular marker technology coming after RFLPs and SSRs [ 46 ]; it has been successfully used to investigate genetic variation among different species and breeds [ 47 — 49 ]. Compared with previous markers, SNPs have the following advantages: 1 they are numerous and widely distributed throughout the entire genome [ 50 ].

Because of their extensive distribution and abundant variations, SNPs play an important role in farm animal population structure, genetic differentiation, origin, and evolution research. For example, linkage disequilibrium LD among different SNPs can be utilized for association analysis. Furthermore, we can gain information concerning animal population diversity and population evolution origins, differentiation, and migrations via SNP haplotypes among different populations.

One disadvantage of SNP markers is the low level information obtained compared with that of a highly polymorphic microsatellite, but this can be compensated for by employing a higher numbers of markers SNP chips and whole-genome sequencing [ 52 , 53 ]. It is the most straight-forward method and provides more complete information on the genetic variation among different populations because it can detect all the variations within the genome.

Currently, the problem with whole-genome sequencing is setting up a high-through data analysis platform to explore useful information for the conservation and utilization of farm animals.

Barcoding is an automatic scanning and identification technology, which has emerged from practical computer technologies. Biological taxonomists apply this principle to species classification, referring to a DNA barcode. The intent of DNA barcoding is to use large-scale screening of one or more reference genes in order to i assign unknown individuals to species, and ii enhance discovery of new species [ 54 , 55 ].

Tautz et al. Subsequently, Hebert et al. Researchers can compile a public library of DNA barcodes linked to named specimens, which can provide a new master key for identifying species diversity [ 57 ]. Compared with time-consuming and inefficient traditional morphological classification [ 58 ], DNA Barcoding has a high accuracy of However, as with the other markers mentioned the DNA barcoding technique also has some disadvantages: 1 the genome fragments are very difficult to obtain and are relatively conservative and have no enough variations.

The above disadvantages can be compensated for by using one or more nuclear gene barcodes together to make a standardized analysis of AnGR. The accurate evaluation of animal genetic resources is the basis for their conservation and utilization. From the first demonstration of RFLPs to the current whole-genome sequencing, many methods have been developed and tested at the DNA sequence level, providing a large number of markers and opening up new opportunities for evaluating diversity in farm animal genetic resources.

With the development of new markers, more accurate genetic evaluation is possible. The development of molecular markers will continue in the near future and provide better understanding of animal genetic resources.

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