Human genome amplification

The development of technically simple and reliable methods to detect sequence variations in specific genes is becoming more important as the number of genes associated with specific diseases grows. Applications of PCR to the human genome include (1) detection of genetic defects associated with inherited diseases (prenatal diagnosis); (2) determination of genetic susceptibility to disease; (3) gene polymorphism detection; (4) determination of the disease risk to offspring in families with affected members; (5) detection of cancer and the extent of residual disease; and (6) the forensic determination of identity [26,103-108]. One advantage of PCR in human genome testing is that only a few cells are required from biopsies, needle aspirations, or paraffin-embedded histologic sections [109]. PCR analysis of gene mutations is now widely used to identify the nature and location of the mutation. Several procedures can be used, such as allelic-specific oligonucleotide hybridization; density gradient electrophoresis; protein truncation test; single-strand conformation polymorphism analysis (sensitivity is 70%-90%); heteroduplex analysis (sensitivity 70%-90%); chemical mismatch cleavage; and RNase cleavage (sensitivity 40%-60%).

The genetic risk of several autoimmune diseases is linked to the major his-tocompatibility complex, with a particular association with certain susceptibility alleles. Identification of the HLA class I or II alleles has been done traditionally by serology in a complement-mediated microcytotoxicity assay. Several reports have compared the results of HLA typing by serology and by restriction fragment length polymorphism, and it has been suggested that 25% or more of alleles may be incorrectly assigned by serology. Moreover, the restriction fragment length polymorphism is more accurate and provides results more quickly (2 hours). The main disadvantages are cost and difficulty of implementation.

Genetic screening and counseling can be offered to couples at risk of bearing children with genetic diseases, or those with predisposing factors (spondyloarthropathy, HLA B27) [110]. Presymptomatic DNA screening is warranted when knowledge of the condition contributes to the health of the individual identified.

In the human genome there are around 10 million single nucleotide polymorphisms. These polymorphisms have been detected in genes responsible of nutrient transport, use, and function (folic acid, iron metabolism, and vitamin D receptor function). Nowadays errors that occur during the amplification process and limit the analysis of genetic alterations can be reduced by the introduction of hairpin-PCR, which separates genuine mutations from errors generated by misincorporation. The method is based on ligation of an artificial DNA hairpin to the DNA target of interest before the PCR.

The PCR (site directed mutagenesis-amplification-created restriction site) has been applied to the diagnosis of cancer and autoimmune diseases by identifying gene mutations in BRACA-1, ras oncogene, b-thalassemia, medium chain acyl coenzyme A dehydrogenase deficiency, hereditary hemo-chromatosis, and ^-antitrypsin deficiency, and by increasing the sensitivity of residual disease detection. The detection of specific p53 mutations in microscopically normal tissue margins is correlated with recurrence; measuring these molecular margins may be useful in determining optimum therapy [111]. By amplifying either tumor-specific DNA abnormalities or tissue-specific mRNA, PCR (or RT-PCR) can detect micrometastases and circulating tumor cells. These and other PCR-based techniques have the potential to improve early detection and pretreatment staging, enhance diagnostic accuracy, identify patients at increased risk for recurrence or metastatic disease, and monitor the effectiveness of therapy. Combining DNA sequencing with PCR analyzes tumor samples rapidly and specifically for minute mutations in relation to clonal expression [112]. Multiplex PCR allows the identification of few neoplastic cells in a million normal cells, in this way providing information of effectiveness of treatment and risk of recurrent disease. RT-PCR has the ability to perform viral load determinations and viral genotyping for oncogenic viruses.

Limitations of polymerase chain reaction

Despite the obvious advantages of these newer procedures (Box 2), there may be potential limitations to DNA amplification technology in the diagnostic microbiology laboratory [113]. The accuracy and reproducibility of PCR assays depend on the technical expertise and experience of the operator. Specificity of the test may be affected by contamination of the specimen during laboratory processing, nonspecific primers being selected for the assay, and PCR conditions not being optimal and allowing nonspecific products to amplify. The most common sources of contamination are from other samples or from previous amplification procedures [114]. Contamination or amplification product carryover of even minute amounts of nucleic acid may result in generation of billions of DNA copies that may lead to false-positive test results [115,116]. For this reason laboratories should have separate

Box 2. Advantages and limitations of PCR


Allows the amplification and detection of minute amounts of nucleic acid sequences in clinical samples High sensitivity High specificity Good reproducibility Versatility

Can provide results within a few hours

Widespread applicability to microbiology and study of infectious diseases Able to analyze gene structure


Potential false-negative results Requires careful interpretation of results A positive test is not validated for all infections Expensive equipment

Cannot be used to study mutational analysis of large genes Technically complex Lack of standardization rooms for different steps of the PCR procedure and must follow stringent quality control measures to prevent contamination or carryover. False-negative test results may occur because of the presence of substances in the specimen that inhibit nucleic acid extraction or amplification.

Certain specimen types (eg, blood) are more likely to contain such inhibitors. The assays may also lack sensitivity if there is a low inoculum of the microorganism present in the clinical specimen. This may be exacerbated if an inadequate sample or very small specimen volume (ie, < 20 mL) is available for testing.

The PCR process relies on primer-mediated DNA replication and it has an absolute requirement for DNA sequence information. Another shortcoming is that the largest DNA fragments that can be reliably amplified are approximately 1000 base pairs in length, and considerably less if the source DNA was extracted from archival tissue blocks (because of partial degradation of the DNA).

PCR is not the tool of choice for performing exhaustive mutational analyses of large human genes that comprise many exons, such as the cystic fi-brosis or breast cancer genes [24]. Interpretation of NAA test results is not always clear-cut. For example, assays may detect the residual DNA of a pathogenic microorganism even after successful treatment, and it is not clear whether this represents the presence of a small number of viable organisms or amplified DNA from nonviable organisms. PCR tests should not be used to monitor the effectiveness of a course of therapy and physicians must be aware of the laboratory testing procedures. In addition, the meaning of a positive PCR test result has not been validated for all infections. For example, it is uncertain whether a positive PCR test result for cytomegalovirus or Chlamydia from a patient's peripheral blood mononuclear cells or synovial fluid or tissue represents active disease, latent infection, or is reactive. Similarly, detection of pneumococcal DNA in blood samples has been reported in asymptomatic children colonized with Streptococcus pneumococ-cus and may not always indicate an invasive infection; however, the presence of viral RNA suggests that ongoing viral replication is occurring. Measuring viral RNA can facilitate the study of mechanisms of viral persistence and initially hidden viral replication. These observations suggest that there is a need for interpretative guidelines based on a correlation of NAA test results with clinical outcome. In such instances, detection of cDNA by RT-PCR of mRNA encoded by the pathogenic organism could be used as evidence of active infection.

The immediate availability of PCR testing in the clinical laboratory has been hindered by the following: (1) the complexity and time-consuming nature of the assays; (2) the need for personnel trained in molecular methods to perform the testing; and (3) the need to define an optimal protocol for each organism and to document the level of sensitivity and specificity of that protocol. Other important problems, such as contamination leading to false-positive reactions, the presence of inhibitors in clinical specimens, and assessing the clinical relevance of positive PCR assays, remain to be resolved [117].

Finally, it must be acknowledged that performance for a PCR assay is generally more expensive than conventional diagnostic laboratory methods. The requirement of separate rooms for pre- and post-PCR steps to reduce the risk of cross-contamination mean that molecular laboratories use an inordinate amount of laboratory space. The cost of these assays has been reported to be high.

Molecular technology involving NAA and detection is a promising tool for the rapid and accurate diagnosis of a variety of infectious diseases and for the confirmation or detection (or both) of antimicrobial resistance. A large number of PCR assays are still under development with the potential to provide accurate and rapid results when conventional methods are either not available, insensitive, or too slow. To date, evaluations of this technology have been generally limited by small samples and have not considered how these assays should fit into routine laboratory procedures, particularly in small, nonreference laboratories. As this technology continues to evolve, it is important to assess the cost effectiveness of these procedures and their real impact on patient management and outcomes.

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