The Human Haplotype Mapping Hap Map Project

Despite the presence of numerous polymorphisms, any two people are 99.9 identical at the DNA sequence level. Understanding the 0.1 difference is important, in part because these differences may be the basis of differences in disease susceptibility and other variations among normal human traits. The key to finding the genetic sources of these variations depends on identification of closely linked markers or landmarks throughout the genome. Genes, RFLPs, VNTRs, STRs, and other genetic structures...

Info

AIDS Society have set viral load levels for initiation of therapy. These levels must be consistently identified in independent laboratories as accurately as possible. Realtime PCR methods offer linear measurement over a wider range than other methods. This precludes the requirement for dilution of high-titer specimens before analysis. The precision or reproducibility of the test is important for establishment of statistically significant differences in the viral load over a serial testing...

Quality Control

Quality control for any clinical laboratory procedure is critical for ensuring the accuracy of patient results, and ensuring the quality of molecular methods in the clinical microbiology laboratory is equally important. The sensitivity of molecular methods is so high that even one molecule of target can be used as a template. Thus, ensuring that the integrity of specimens is maintained, i.e., that specimens are not contaminated by other specimens or with the products of previous amplification...

Documentation of Test Results

Test results in the form of electropherograms, gel images, and autoradiograms should be of sufficiently high quality that results are unequivocal. This includes clear bands or peaks without high background, cross hybridization, distortions, and other artifacts. Controls should also be clear and consistent and reflect the expected size or level. Molecular weight ladders on gels, autoradiograms, or electropherograms should cover the expected range of band or peak sizes produced from the specimen....

Engraftment Testing Using DNA Polymorphisms

Bone marrow transplantation is a method used to treat malignant and nonmalignant blood disorders, as well as some solid tumors. The transplant approach can be autologous (from self), in which cells from the patient's own bone marrow are removed and stored. The patient then receives high doses of chemotherapy and or radiotherapy. The portion of marrow previously removed from the patient may also be purged of cancer cells before being returned to the patient. Alternatively, allogeneic transplants...

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Lela Buckingham

Results because the organism can be found as normal flora in some patient populations 4. Which of the following controls are critical for ensuring that amplification is occurring in a patient sample and that the lack of PCR product is not due to the presence of inhibitors 5. A PCR assay performed to detect Bordetella pertussis on sputum obtained from a 14-year-old girl who has had a chronic cough had two bands, one consistent with the internal control and the other consistent with the size...

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Ribosomal, 58-59, 60f Paired box-Forkhead in rhabdomyosarcoma, 340-341 Palindromic defined, 13 Panel reactive antibodies in transplant screening, 386 Parainfluenza viruses detection of, 294t Parasites detection and identification of, 300 Parentage testing restriction fragment length polymorphisms in, 22, 229f short tandem repeats in, 238-240, 239b-240b, 239f, 239t Parvovirus B19 detection of, 293t Paternal lineage testing Y-short tandem repeats in, 241, 244-245 Paternity probability equation...

Advanced Concepts

More detailed crossmatch information is achieved by separate analysis of B and T donor lymphocytes. Unactivated T cells display class I antigens, and B cells display both class I and class II antigens. Therefore, if B cells cross react with the serum antibodies and T cells do not, the serum antibodies are likely against class II antigens. Figure 15-7 Detection of serum antibodies using bead arrays. In this illustration, separate preparations of beads are conjugated to two different known...

Mpsgcwgcwr

Express these mutations using the accepted nomenclature. 1. Yeung A, Hattangadi D, Blakesley L, et al. Enzymatic mutation detection technologies. BioTechniques 2005 38(5) 749-58. 2. Orita M, Iwahana H, Kanazawa H, et al. Detection of polymorphisms of human DNA by gel elec-trophoresis as single-strand conformation polymorphisms. Proceedings of the National Academy of Sciences 1989 86 2766-70. 3. Orita M, Suzuki Y, Sekiya T, et al. Rapid and sensitive detection of point mutations and DNA...

Specimen Handling

Molecular tests, like any clinical laboratory tests, require optimal specimen handling and processing for accurate and consistent test results. The success of a test procedure is affected by the age, type, and condition of specimens. Therefore, specimen collection, transport, and handling in the laboratory require careful attention. Preanalytical variables, both controllable and uncontrollable, must be taken into account for proper interpretation of test results. Preanalytical error is the...

L V D J C

Figure 14-15 Immunoglobulin heavy chain gene on chromosome 14 consists of a series of variable (V), diversity (D), and joining (J) gene segments (germline configuration). The V segments are accompanied by a short leader region (L). One of each type of segment, V, D, and J, is selected and combined by an intrachromosomal recombination event, first D and J, and then V and D. The C (constant) segments are joined through splicing or a secondary recombination event, class switching. the Dh segment....

Buffer Additives

Buffer additives are often used to modify sample molecules in ways that affect their migration. Examples of these additives are formamide, urea, and various detergents. Denaturing agents, such as formamide or urea, break hydrogen bonds between complementary strands or within the same strand of DNA or RNA. The conformation or solubility of molecules can be standardized by the addition of one or both of these agents. Formamide and heat added to DNA and RNA break and block the hydrogen bonding...

Chapter

Laboratory tests include electron microscopy, serology, and RT-PCR. Electron microscopy and immune electron microscopy require specialized equipment and technical expertise. Detection of serum antibodies to the virus is not always straightforward, as most of the adult (but not child) population has serum antibodies to this virus. Furthermore, it can take several days after exposure to develop detectable IgM antibodies. RT-PCR, therefore, is the method of choice for...

Crude Lysis

Although high-quality DNA preparations are tantamount to successful procedures, there are circumstances that either preclude or prohibit extensive DNA purification. These include screening large numbers of samples by simple methods (e.g., electrophoresis with or without restriction enzyme digestion and some amplification procedures), isolation of DNA from limited amounts of starting material, and isolation of DNA from challenging samples such as fixed, paraffin-embedded tissues. In these cases,...

Instrument Maintenance

Instruments used in the molecular laboratory must be monitored and maintained for consistent performance and accurate test results. Manufacturers supply recommendations for routine maintenance. Service contracts are used to provide support from trained service technicians. Laboratory professionals maintain a schedule and instructions for all routine maintenance, such as checking temperature settings, timers, and background levels. Parts are replaced as required or specified by the instrument...

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Rhoeae and C. trachomatis have been considered the gold standard, but when compared with nucleic acid amplification assays, the sensitivity of culture for N. gonorrhoeae is 85 -100 and for C. trachomatis 80 .9 Nucleic acid amplification assays have the additional advantages of being rapid, and testing can be batched and automated, resulting in further savings for the laboratory. The first molecular-based assay available for N. gonorrhoeae and C. trachomatis was AccuProbe from Gen- Probe, Inc....

Is 481

Pertussis toxin promoter region DNA polymerase gene plyA (pneumolysin) lytA (autolysin) pbp2a (penicillin-binding protein) pbp2b pspA (pneumococcal surface protein) 16S rRNA M. tuberculosis is an important cause of respiratory tract infections, is a worldwide problem, and results in infections that have significant morbidity and mortality levels. The diagnosis of tuberculosis can be difficult and can take prolonged periods during which the patient is not adequately treated and may spread the...

F13a01

An engraftment analysis was performed by capillary gel electrophoresis and fluorescence detection. The fluorescence as measured by the instrument under the FESFPS donor peak was 28118 units, and that under the FESFPS recipient peak was 72691. What is the percent donor in this specimen 7. The T-cell fraction from the blood sample in Question 6 was separated and measured for donor cells. Analysis of the FESFPS locus in the T-cell fraction yielded 15362 fluorescence units under the donor peak and...

Molecular Strain Typing Methods for Epidemiological Studies

In community or clinical settings, when the same organism is isolated multiple times, whether in the same patient or from different patients, the physician wants to know if the isolates were independently acquired, i.e., came from different sources, or if they came from the same source. With this knowledge, the physician can act to control the transmission of the organism, especially if it is being transmitted from a common source and that source has been identified. Most of the time, these...

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R factors carry resistance to common antibiotics such as chloramphenicol, tetracy-cline, ampicillin, and streptomycin. Another class of plasmid, colicinogenic factors, carries resistance to bac-teriocins, toxic proteins manufactured by bacteria. Plasmids can replicate in the host cell but cannot survive outside of the cell as viruses do. The acquisition of the resistance genes from host chromosomes of unknown bacteria is the presumed origin of these resistance...