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        Atom Sciences has been notified by the National Institutes of Health that ASI will receive funding to develop a new highly selective DNA sequence detection method to detect and distinguish among members of the genus Mycobacterium (see Press Release). The new technique is called Limited Primer Extension (LPE) and it targets sequences that lack one of the four nucleosides over a span of 12 to 20 bases. Polymerase-driven primer extension across this span of DNA sequence is terminated at the end of the span by incorporation of the dideoxy form of the nucleotide that is complementary to the base absent within the span. This forms short, single-stranded products that are ideally suited for oligodeoxyribonucleotide (ODN) microarray hybridization. 

Tuberculosis - Tuberculosis (TB) remains a leading cause of death worldwide, despite a greatly increased understanding of its etiology and advances in therapy options and disease diagnosis; approximately two million die of TB annually worldwide [1]. Although TB is a less significant killer in the US than in many other parts of the world, there were still 14,871 TB cases reported in the US in 2003 [2]. The increasing incidence of TB strains exhibiting multiple drug resistance, and the AIDS epidemic have exacerbated efforts to fight TB [1].

TB is caused by Mycobacterium tuberculosis (MTB), which is a Gram-positive, acid-fast bacterium that is readily isolated from many environmental sources, including soil and water. TB is highly contagious, being readily spread via respiratory aerosols, which contributes significantly to the costs of managing TB patients because they must be kept in isolation for long periods. Another problematic aspect of TB management is that standard diagnostic tests take several weeks to complete because they involve growth on selective media and this organism grows very slowly in vitro. Rapid liquid culture systems have reduced this time to 7 to 12 days requiring only 10 – 100 bacilli per milliliter of sputum [3]. While a positive TB diagnosis based on in vitro cultures can be achieved within 2-3 weeks, cultures that are negative must be kept for up to 8 weeks before a reliable negative result can be determined. Patients with negative results (including a large proportion of AIDS patients [4]) are often infected with other species of Mycobacterium - usually those of the M. avium complex (MAC; M. avium, M. intracellulare, and other unclassified Mycobacterium species.

Present Methods - Conventional techniques for detecting TB included acid-fast smearing and culturing. The acid-fast smearing lacks sensitivity and cannot distinguish between MTB and other bacilli. The culture technique is sensitive and specific but typically requires 3-6 weeks to complete [5]. There are currently at least two effective and rapid diagnostic kits available commercially for MTB detection in clinical specimens (Roche Amplicor, GenProbe E-MTB) [6]. Both tests employ molecular approaches to detect MTB-specific nucleic acids and can be completed in less than one day, allowing patients to be moved out of expensive isolation facilities immediately upon receipt of a negative TB test result. However, while both tests yield results that approach the sensitivity and specificity of in vitro culture tests [1], neither identifies the non-MTB pathogen causing the TB-like symptoms and thus offer no benefit to those patients with negative TB test results. Presently, additional in vitro culture tests are needed to diagnose infections by these alternate pathogens, which are often species of Mycobacterium other than tuberculosis. A new technique has been reported by researchers at Nanyang Technological University that uses lasers and claims to reduce the analysis for TB to days instead of weeks [5]. However, it appears that this method still provides no information about non-MTB pathogens that produce TB-like symptoms. The current “Gold Standard” for molecular diagnostic discrimination among species of the Mycobacterium genus and for molecular identification of antimicrobial resistance is sequencing [1 ,6]. Using genus-specific primers to generate sequencing templates with the PCR, all of the currently recognized mycobacteria species can be discriminated. Nevertheless, sequencing is not commonly used in molecular diagnostic practice due to cost and technical requirements that include laboratory instrumentation and the number of independent hands-on steps required. 

Therefore, an ideal TB diagnostic test would not only detect MTB rapidly (hours) and with great sensitivity and accuracy, but it would simultaneously test for and identify a cadre of related organisms that are commonly associated with TB-like symptoms. Such a multiplexed approach to TB testing is the subject of the ASI research.

How the New Technology Works - The LPE process is based on the fact that natural DNA has a high frequency of nucleotide sequence stretches that lack one of the 4 bases. Figure 1A shows an example of a 16-base stretch of DNA lacking adenine. We call this the void region and it is flanked on both sides by the missing base, in this case, adenine. A primer is designed to anneal to the DNA adjacent to the void region on the 3' side. Then a thermal-stable polymerase uses standard deoxyribonucleoside triphosphates (dNTPs) to extend the primer using the void region as template. However, once the extension reaction reaches the missing base at the 5' end of the void region, a dye-labeled dideoxyribonucleoside triphosphate (ddNTP) is incorporated into the extension. ddNTPs do not support the extension reaction and so the extension product terminates at this point. The full extension product includes the  primer and the extension, the portion made by the extension reaction. The extension product will always terminate in the complement of the missing base in the void region. The primer extension product can be dissociated from the template using temperature or proprietary methods and the process can be repeated, providing linear amplification of the extension product. 

Figure 1B shows the next step in the process. A probe that is complementary to a section of the extension product is tethered to a DNA microarray surface via an appropriate linker. The probe may have the same sequence as the void region, as depicted in Figure 1B, or the length of the probe can be varied by shortening to be a fraction of the void region sequence or by lengthening to include a few bases adjacent to the void region on the 3' end. In this way, the T_d of the probe/extension product hybrid can be adjusted. 

Advantages of LPE - The LPE process creates short, single-stranded targets that are ideal for hybridization on a DNA microarray. In addition, the capacity for multiplexing is not limited by chain reaction products as in the PCR because chain elongation is terminated early. Furthermore, non-targeted amplicons derived from mis-priming events, unintended amplicons, primer-dimer formation, and other sources are not propagated as it is in the PCR. Therefore LPE can be extensively multiplexed, making it capable of producing symptom-based diagnostics that identify an active pathogen among dozens or even hundreds of candidates. This property suggests that LPE techniques would be invaluable as a tool to quickly identify a bioterrorist threat or an outbreak of disease. 

5.     http://www.news-medical.net/?id=2009

6.     Soini, H., and J. M. Musser. 2001. Molecular diagnosis of mycobacteria. Clinical Chem. 47:809-814.

Research funded by the National Institutes of Health under Small Business Innovation Research Grant 1 R43 AI064969-01. Data and comments are the sole responsibility of Atom Sciences, Inc. and do not necessarily reflect the views of NIH.

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