Wendy Chao and Dr. Richard Robison, Microbiology
After over a century of decline, tuberculosis (TB) has re-emerged as the leading killer among infectious diseases. In addition, Mycobacterium tuberculosis, the causative agent of TB, has developed strains resistant to multiple antibiotics, making the disease increasingly difficult to treat.
Rapid diagnosis is critical in treating multidrug-resistant TB (MDRTB). Molecular techniques based on polymerase chain reaction (PCR) are rapidly replacing traditional culture methods of diagnosing MDRTB. Random amplification of polymorphic DNA (RAPD), a technique commonly used to identify unique DNA, can potentially identify drug resistance markers in M. tuberculosis. Obtaining the DNA sequences of these markers will aid the construction of diagnostic DNA probes, which can rapidly identify drug resistance in clinical TB isolates.
For the past year, I have been developing efficient ways to sequence various RAPD markers using the LI-COR 4000L Automated DNA Sequencing System. This automated system uses fluorescent-labeled DNA primers, which are detected by infrared laser. I have been able to successfully sequence DNA using cycle labeling and sequencing (CLS). In this method of DNA sequencing, fluorescent labels are incorporated into our own custom-designed primers, which eased the time and cost of obtaining pre-labeled primers.
This project took an interesting turn when PCR was used to confirm RAPD markers of mouse Y chromosomes. Researchers in our department have previously reported RAPD markers that are gender- and strain-specific in mice (1,2). Other laboratories have also detected polymorphisms in mouse DNA using RAPD (3). However, PCR has revealed that some of these markers are neither gender-specific nor strain-specific, despite showing specificity in RAPD. I have been sequencing these PCR markers to confirm these findings, and sequence data will be compiled into an article to be submitted for peer review and publication next month. These findings question the reliability of RAPD for finding DNA markers, and suggest that PCR should be used in place of, or at least to confirm, the RAPD technique.
After concluding this project, I will continue sequencing drug-resistance markers in clinical TB isolates using automated sequencing and CLS. The DNA sequences obtained will be submitted to electronic databases containing known DNA sequences, which can be accessed for diagnostic comparisons and epidemiological studies.
References
- Woodward, S. R., Sudweeks, J., Teuscher, C. (I 993). Random sequence oligonucleotide primers detect polymorphic DNA products which segregate in inbred strains of mice. Mammalian Genome 3, 73-78.
- Wardell, B. B. et al. (1992). The identification of Y Chromosome-linked markers with
random sequence oligonucleotide primers. Mammalian Genome 4: 109-112. - Cheah, Y.-C. et al. (1994). New murine polymorphisms detected by random amplified
polymorphic DNA (RAPD) PCR and mapped by use of recombinant inbred strains. Mammalian Genome 5, 762-767.