In the context of RFLP analysis, these probes allow scientists to "see" specific DNA fragments among the thousands produced when a genome is cut by restriction enzymes.
1. How RFLP Probes Work
The RFLP process relies on the fact that different individuals have slight differences in their DNA sequences (polymorphisms). These differences can change where a restriction enzyme cuts the DNA.
- Digestion: DNA is cut into fragments by restriction enzymes.
- Electrophoresis: The fragments are separated by size on a gel.
- Southern Blotting: The DNA is transferred from the gel to a nylon membrane.
- Hybridization (The Probe's Job): The membrane is exposed to the RFLP probe. The probe "hunts" for its exact matching sequence on the membrane and binds (hybridizes) to it.
- Visualization: Because the probe is labeled (usually with radioactivity or a fluorescent dye), a researcher can see exactly where the probe bound, revealing the size of the specific fragments.
2. Types of RFLP Probes
- cDNA Probes: Derived from complementary DNA (mRNA converted back to DNA). These represent the coding regions (exons) of the genome.
- Genomic DNA Probes: Derived from random segments of the genome, which may include non-coding regions.
- Single-locus Probes: Bind to only one location in the genome, producing one or two bands. These are easier to interpret.
- Multi-locus Probes: Bind to multiple repetitive sequences across the genome, producing a complex "barcode" or fingerprint.
3. Characteristics of a Good Probe
- Specificity: It must only bind to the target sequence.
- Labeling: It must be tagged (with $^{32}P$ radioactive phosphorus or non-radioactive biotin/chemiluminescence) so it can be detected on X-ray film.
- Length: Usually range from 500 to several thousand base pairs.
4. Applications
Historically, RFLP probes were the gold standard for several fields: Paternity Testing: Comparing the fragment sizes of a child with those of the potential parents. Forensics: DNA fingerprinting to identify suspects (the "original" DNA test used in the 1980s and 90s). Genetic Mapping: Locating genes responsible for hereditary diseases (e.g., Cystic Fibrosis, Sickle Cell Anemia). Evolutionary Biology: Studying genetic diversity and relationships between different species.
5. Advantages and Disadvantages
| Advantages | Disadvantages |
|---|---|
| Highly Accurate: Very reliable for identifying genetic relationships. | Requires a lot of DNA: You need a relatively large, high-quality sample. |
| Co-dominant Markers: Can distinguish between homozygous and heterozygous individuals. | Slow and Laborious: The process takes several days to weeks. |
| Consistent: Results are highly reproducible across different labs. | Radioactivity: Historically required hazardous radioactive materials. |
6. Modern Status
While RFLP probes were revolutionary, they have been largely replaced by PCR (Polymerase Chain Reaction) based methods, such as STRs (Short Tandem Repeats) and SNPs (Single Nucleotide Polymorphisms).
PCR-based methods are preferred today because they are much faster, cheaper, and can work with tiny or degraded DNA samples (like a single drop of blood or an old bone), which RFLP probes cannot do effectively.