Although restriction enzymes are widely used in molecular cloning, their use as molecular tools extends to other common applications in molecular biology. Two important applications are DNA fingerprinting and methylation analysis, which are methods to map sequences and analyze epigenetic patterns in the genome.
The concept of DNA fingerprinting or profiling arose in the 1980s as a means to genetically identify individuals based on unique patterns of DNA fragment sizes generated from their genomes. In other words, the DNA fragments and their length variations could be used as differentiating markers, or “fingerprints”, for genetic identification (i.e., of alleles) instead of relying solely on phenotypic characteristics.
Today the use of DNA fingerprinting techniques is very common. While much of the general public is aware of its significance in forensics and criminal cases, DNA fingerprinting and mapping have broad applications in other areas such as disease testing and plant breeding. The extensive use of DNA fingerprinting has led to the development of numerous DNA fingerprinting methods, with the choice of method primarily depending on the experimental goals and the study organism(s).
Restriction fragment length polymorphism (RFLP)
Restriction fragment length polymorphism, or RFLP (pronounced “rif-lip”), is the basis for one of the oldest DNA fingerprinting methods. The typical workflow of this method involves restriction digestion, fragment separation, Southern blotting, probe hybridization, and visualization (Figure 1).
|Figure 1. Basic workflow for identifying restriction fragment length polymorphisms (RFLPs).|
In the first step, purified genomic DNA is digested with one or more restriction enzymes. The choice of restriction enzymes is usually based on the ability to distinguish genetic variability and the cost of the enzymes. The digested fragments are separated by gel electrophoresis and appear as a continuous smear on the gel due to the broad distribution of fragment sizes generated by the enzymes.
To detect the desired fragments, the gel-separated DNA fragments are transferred to a nitrocellulose or PVDF membrane for handling and detection. A labeled single-stranded DNA probe is hybridized to the membrane to identify a subset of fragments. The results are visualized to reveal the unique RFLP fingerprint.
Probes for RLFPs are based on single- to low-copy number sequences in a genome and usually range between 500 and 2,000 bases. Probes are labeled to detect even low amounts of samples and identify the fragments that will become the basis of the fingerprint. The resulting RFLP markers observed are a result of specific probe and restriction enzyme combinations. For example, Probe A and EcoRI-digested genomic DNA will define one RFLP for a specific genome. Probe A and HindIII-digested DNA will define a different RFLP for that genome (Figure 2A). Knowledge of the template sequence, though not required, allows faster development of useful RFLP probes.
RFLPs and restriction enzymes can also be used to detect DNA differences between two individuals. Figure 2B illustrates probe hybridization and detection on a simplistic level, comparing two individuals for HindIII-based RFLPs of two alleles (labeled “1” and “2”). In this example, probe A detects different restriction fingerprints in the two individuals due to loss or gain of a HindIII restriction site on allele 2. In most cases, however, fragment length variability between individuals is a result of insertion or deletion of DNA sequences outside of the restriction sites, caused by natural recombination and replication. RFLP analysis is also used in applications such as genetic counseling, plant and animal breeding programs, and disease monitoring.
Despite its usefulness, RFLP analysis has some limitations. The analysis requires a large amount of starting sample DNA, and the entire process is slow and cumbersome. With the development of PCR, many of these drawbacks have been addressed—for example, with amplification fragment length polymorphism (AFLP), which requires far less sample and can be performed using more rapid PCR-based protocols.
AFLP is another genetic mapping technique that relies on RFLP followed by selective PCR to generate amplified fragments from genomic DNA of any organism, without prior knowledge of the genomic sequence. In addition, AFLP analysis requires only small amounts of starting template (typically nanograms).
While AFLP was first developed for plant studies , it is now used for a variety of applications, such as:
- Creation of genetic maps for new species
- Determination of relatedness among cultivars
- Establishment of linkage groups in crosses
- Studies of genetic diversity and molecular phylogeny
The AFLP procedure involves digestion of genomic DNA to produce a population of restriction fragments, ligation of priming site adaptors, amplification by PCR, separation of fragments by electrophoresis, and finally visualization of PCR amplicons by either autoradiography or fluorescence (Figure 3).
In the first step of AFLP, genomic DNA samples are enzymatically digested, typically with EcoRI and MseI. The GC content of the genomic DNA is relevant to the effectiveness of digestion and the resulting fingerprint. For example, optimal digestion with MseI is obtained when the GC content is <50%. High GC content (e.g., >65%) may hinder MseI digestion and result in a low quantity of fragments. Similarly, low GC content favors more complete digestion by EcoRI.
After digestion is complete, two adaptors are ligated to the ends of the fragments. The design of these adaptors includes an additional 19–22 bp to allow subsequent primer binding, and their ligation results in the loss of the original MseI and EcoRI recognition sites (Figure 3B).
Next, two rounds of PCR amplification are carried out, one that is pre-selective followed by a more selective reaction. During the pre-selective PCR, DNA fragments with ligated EcoRI and MseI adaptors are amplified (Figure 3C). After this step, a more selective PCR is performed with primer sets that carry up to three additional bases at the 3′ end of the primer (Figure 3D). These primers include selective nucleotides that, in combination with stringent PCR conditions, help ensure that amplification occurs only for those DNA fragments that share sequence homology with the 3′ ends of the selective primers. In the selective amplification step, the primers to the EcoRI adaptors are either radioactively or fluorescently labeled for downstream detection of fragments, whereas MseI primers are unlabeled.
The combination of selective primers and selective labeling refines the subsequent map to reveal a unique fingerprint by gel or capillary electrophoresis (Figure 3E). The optimal range for a fingerprint after amplification is between 50 and 200 fragments, ranging from 45 to 500 nucleotides in length. Of the visualization methods, fluorescent labeling in combination with capillary gel electrophoresis is preferred, since the process can be automated from fragment separation to data collection for efficiency and robustness.