Verification Of Pcr Products

Although DNA bands of the expected size on an agarose or polyacrylamide gel are an encouraging sign that the reaction produced the desired amplicons, the identity of the PCR product should be verified by a secondary method. Verification is usually accomplished by nucleotide sequencing, restriction mapping, or sequence-specific probe hybridization (Fig. 3). Sequencing a PCR product provides the best information on the identity of the amplicon and represents the best method for verification of...

Conclusion

This chapter was intended to provide the reader with some of the basic concepts of molecular genetics. One should keep in mind not only the applications of this technology, which will be discussed in the following chapters, but also the nonscience consequences that arise as a result of genetic disease testing. Nucleic acid-based laboratory testing can be performed as a diagnostic procedure, for carrier testing, as a prenatal diagnostic test, or for presymptomatic and susceptibility testing...

Clinical Validation

Clinical validations are intended to show that the results produced by an assay correlate with clinical disease. Neither CLIA '88 nor CAP requires the clinical validation of an assay prior to implementation of its use in a testing laboratory. Although clinical validations are not required before testing, these types of validation study are still needed before the assay can be used for clinical diagnosis. In many instances, clinical studies for a given analyte have been performed and published...

Electrophoretic Separation Of Rna A

Protocol for Northern blot analysis is provided in Table 3. When analyzing total cellular RNA, the quantity of RNA loaded onto the gel can range from 5 to 30 g. The ideal concentration should be determined empirically and depends on the quantity and quality of target mRNA as well as specificity of the probe. Analysis of purified mRNA can be performed with as little as 1.0 g, although a greater quantity (5-10 g) is typically used to permit multiple uses of a single membrane. Just prior to...

Analysis Of Pcr Products

There are numerous methods for analysis of PCR products (Figs. 2 and 3). The method of choice for analysis of PCR products will depend on the type of information that is desired. Typical analysis of PCR products will involve electrophoretic separation of amplicons and visualization with ethidium bromide or other DNA dye. In most cases, amplification products can be analyzed using standard agarose gel electrophoresis. Agarose gel electrophoresis effectively separates DNA products over a wide...

Genomic Instability In Human Cancer

An appropriate definition of genomic instability is needed before a complete understanding of the interconnecting causes and consequences of genomic instability can be developed, and the contribution of this phenomenon to neoplastic transformation can be appreciated. The observation that most cancer cells contain discernible genetic abnormalities (chromosomal aberrations and or DNA sequence abnormalities) suggests that all neoplastically transformed cells have sustained genetic damage and might...

Trinucleotide Repeat Measurements

Basic research and technological advances in human genetics, biochemistry, and model systems have brought much progress toward the understanding of human infectious, hereditary, and somatically acquired diseases. In fact, whereas in 1991, triplet repeat expansion diseases could be described in a single article, now the remarkable developments within each disease has created volumes of work (4,5). One factor contributing to triplet repeat diseases is the adoption of unusual non-B DNA structures...

Genes Of Thrombotic Or Thrombolytic Function

FACTORS V AND II The backround on hemostasis, molecular mechanisms for venous thrombosis, and description of the common mutations for Factors V and II are given in Chapter 41. Given the important role of Factor V Leiden in venous thrombosis, studies were conducted to determine if this polymorphism is a risk factor for arterial thrombosis. Surprisingly, the data have consistently shown that the presence of Factor V Leiden is not associated with an increased incidence of coronary artery...

Early Modern Medicine

Chemistry as a science was developing rapidly in the 18 th and early 19th centuries. Diabetes was the subject of intensive investigation by the medical chemists. Physicians like John Rollo and William Cruikshank demonstrated the presence or absence of sugar in the urine of diabetics, depending on the state of the patient's disease (5). They were proponents of being able to monitor a patient's status by knowing if there was sugar in the patient's urine (5). The conventional wisdom of the time...

Introduction

A framework for ensuring laboratory quality was laid down by Congress in the Clinical Laboratory Improvement Act of 1967. Because of public concerns about the quality of clinical laboratory testing in the United States, Congress passed the Clinical Laboratory Improvement Amendments of 1988 (CLIA'88) setting forth uniform quality standards for all laboratories performing tests for health purposes on human specimens. Laboratories must register with the Department of Health and Human Services...

Optimization Of Pcr Reactions

A number of different factors can significantly impact on PCR sensitivity and specificity, including (1) oligonucleotide primer design, (2) PCR cycling parameters (number of cycles, cycle times, and temperatures), and (3) the composition of the PCR mixture (Mg2+ concentration). For most PCR applications, the most critical parameter is the annealing temperature for the oligonucleotide primers employed. The maximum annealing temperature is determined by the primer with the lowest Tm. Exceeding...

Oligonucleotide Ligation Assay

Oligonucleotide primers that which have annealed to adjacent sites on single-stranded DNA, such that there is no gap between them, can be covalently joined together by DNA ligase. Fig. 3. Multiplex ligation-dependent probe amplification. Fig. 3. Multiplex ligation-dependent probe amplification. This is the basis for the oligoligation assay (oligonucleotide ligation OLA ). Successive cycles of denaturation, annealing, and ligation result in a linear amplification of the target sequence. In...

Troubleshooting Northern Blots

Some genes are simply not expressed at any level in certain cell types. However, if a known positive control was included on the gel and no positive signal was produced, there could be a technical problem. Consult the following checklist to eliminate some possibilities Was the RNA of good quality (integrity gel, ethidium bromide staining) Was electrophoresis of the RNA sufficient (migration of RNA bp ladder) Was the RNA transferred to the nylon membrane (stain the Was hybridization time...

Info

Standards provide a reliable way to both monitor the sensitivity of mutation detection and to discern possible introduction of errors that potentially occur during the amplification and separation procedures (16-18). Mutations in the TP53 gene are the single most common genetic alterations observed in human cancers (19,20). For this reason, mutation detection of TP53 was selected as a candidate by NIST to create standards. These are expected to assist health care industries...

Neurofibromatosis

Neurofibromatosis 1 (NF 1) is an autosomal dominant disorder affecting approx 1 3500 individuals. It is characterized by tumors of neural crest origin. Predominant manifestations include cafe au lait spots and cutaneous or subcutaneous neu-rofibromas (benign tumors of peripheral nerves). Other findings include axillary freckling, Lisch nodules (iris hamartomas), sco-liosis, plexiform neurofibromas, macrocephaly, short stature, seizures, and localized hypertrophy. Over half of individuals report...

Reportable Range

The reportable range of an assay is the range of values that will generate a positive result for the specimens assayed by the test procedure. Clinical patient specimens, positive for the given nucleic acid analyte, are assayed to determine the values expected to be produced by clinically positive specimens. Depending on the type of assay (qualitative, quantitative, analytical), the reportable range can have multiple definitions. For qualitative tests that are only detecting the presence of a...

Pharmacogenetics In Drug Development

Pharmacogenetics has without doubt become a primary field of research, with extensive involvement of both academic labs and industry. Pharmaceutical companies have invested heavily in genomics-era technologies, and many routinely include pharmacogenetics in their clinical studies (64). Some companies make strategic decisions to investigate pharmacogenetics in specific phases of drug development, but increasingly companies are electing to include pharmacogenetics in all phases of their clinical...

The Southern Blot

The DNA blot was developed by Southern in 1975 (3), and it remains the method of choice among many researchers for From Molecular Diagnostics For the Clinical Laboratorian, Second Edition Edited by W. B. Coleman and G. J. Tsongalis Humana Press Inc., Totowa, NJ reliable, quantitative detection of specific DNA sequences. The Southern blot can detect the presence of homologous genes across species. For example, if a biologically relevant gene has been located in the rat, it is possible to...

Genetics

REPEAT INSTABILITY Each of the above-described disorders is characterized by the presence of a trinucleotide repeat within the gene responsible for that disorder. For fragile X syndrome and SCA12, the trinucleotide repeat is localized to the 5' untranslated region and for DM I and SCA8 it is present in the 3' untranslated region. For SBMA, HD, SCA1, SCA2, SCA3 MJD, SCA6, SCA7, and DRPLA, the repeat is within the coding region. In 96 of patients with Friedreich ataxia, the repeat is located...

An Overview Of Dna Methylation In Health And Disease

INTRODUCTION Although assessing DNA methy-lation has not yet become commonplace in the diagnostic molecular pathology laboratory, many tests involving methyla-tion analysis will become part of routine practice. In this chapter, a practical approach will be taken toward evaluating DNA methylation. The principal question likely to be asked in the pathology laboratory is whether a specific gene or region is methylated in a specific pathological situation for example, is the MLH1 gene promoter...

Dna Function

DNA serves two important functions with respect to cellular homeostasis the storage of genetic information and the transmission of genetic information. In order to fulfill both of these functions, the DNA molecule must serve as a template. The cellular DNA provides the source of information for the synthesis of all the proteins in the cell. In this respect, DNA serves as a template for the synthesis of RNA. In cell division, DNA serves as the source of information inherited by progeny cells. In...

Nucleic Acid Blotting Techniques

Terry Amiss and Sharon Collins Presnell This chapter deals with basic concepts and techniques in nucleic acid blotting. Many of the techniques involved with Southern blotting and Northern blotting are similar. Negatively charged, purified nucleic acids from prokaryotic or eukaryotic cells are separated according to size by electrophoresis through an agarose gel matrix. The RNA or denatured DNA is subsequently transferred and immobilized onto a membrane composed of nitrocellulose or nylon. The...

Methodology Of Southern Blot Analysis

PREPARATION OF DNA FOR SOUTHERN BLOTTING Most basic techniques for purification of DNA produce material appropriate for Southern analysis. Standard Southern blot protocols recommend the use of 10 g of DNA when analyzing single-copy genes (6). However, when the amount of DNA is limiting, smaller quantities can be used without compromising the signal by altering the geometry of the sample well during electrophoresis (decreasing the width of the well will increase the intensity of the final...

Conclusions

SPECIFICITY VS SENSITIVITY Developing testing methodologies that are sensitive and specific can be difficult. Linear primers or probes complementary toward specific areas of the nucleic acid target can result in false-negative results if the target region is polymorphic. These polymorphisms obstruct base-pairing with the primer or probe. Inaccurate quantification resulting from these variations has been demonstrated from studies with human immunodeficiency virus (HIV) (46). These variants...

Enzymaticbased Signal Amplification

Another category of signal amplification assays utilizes enzymatic reactions that can detect conformation changes in a target. The most frequently used method in this category utilizes the Cleavase enzyme, which can detect single-basepair changes in the Invader assay (Third Wave Technologies, Madison, WI) (43,44) using linear probes, Whereas another method (RAM) combines RCA and strand displacement amplification to amplify a circular DNA probe that serves as the reporter molecule (45). 3.2.1....

Signal Amplification

Signal amplification methods have been available in serology for many years and are characterized by increases in the detection of a target molecule rather than the molecule itself. Similar principles are used when detecting various nucleic acid targets. Two approaches of signal amplification methods involve nonenzymatic or enzymatic technologies. In nonenzy-matic signal amplification, the target is immobilized or localized using complementary probes. Ultimately, the hybrid molecule is detected...

Chromosomal Abnormalities In Cancer

The majority of human cancers (including solid tumors, leukemias, and lymphomas) contain chromosomal abnormalities, consisting of either numerical changes (aneuploidy) and or structural aberrations (102,103). These two general types of chromosomal damage might reflect two distinct mechanisms of chromosomal instability (9,104) (1) chromosome number instability and (2) chromosome structure instability. In some forms of cancer, chromosomal instabilities predominate over nucleotide sequence...

Categories Of Genetic Disorders

Currently, there is an enormous amount of information with respect to numbers of and characteristics of various genetic Fig. 1. A human male karyotype (G-banded) showing the 22 pairs of autosomes and the 2 sex chromosomes (XY). Fig. 1. A human male karyotype (G-banded) showing the 22 pairs of autosomes and the 2 sex chromosomes (XY). Types of Noncoding Junk DNA Sequence Intron DNA sequences that interrupt the coding (exon) sequences of a gene Satellite Short repetitive DNA sequences that occur...

Cd4apc

Peripheral blood specimen, gated on CD3+ T-cells. In this manner, the percentage of CD4+ T-cells (lower right quadrant) and CD8+ T-cells (upper left quadrant) can be quantified. including disorders of cellular immunity (11). Lymphocyte subset analysis by flow cytometry is typically one of the first laboratory tests performed in the clinical evaluation of such patients. Moreover, lymphocyte activation, cytokine expression within specific cell subsets, and cellular cytotoxicity can all be...

Sample Selection

In addition to the RNA isolation techniques discussed in Section 3.2, there are a number of sample selection issues key to the proper interpretation of microarray assays (27). Sample selection includes considerations of the population of patients selected for the study, the selection of tissue within the patient, and method of selecting cells for RNA collection. The proper selection of patients cannot be overemphasized because no analytical plan can overcome a major error in sample selection....

How Mutations Affect The Endocrine System

All endocrine systems consist of interacting networks of lig-ands, receptors, postreceptor signaling molecules, enzymes, and protein products. Each of these proteins is encoded by a gene, and these genes, in turn, are regulated by networks of transcription factors. Mutations in any of the involved genes can lead to alterations in the function of a given system and, thus, to disease. Selected endocrine disorders that illustrate particular molecular mechanisms of disease will be discussed here...

Abnormal Dna Repair Contributes To Genomic Instability And Cancer Predisposition

The ability to repair damaged DNA is fundamental to all biological processes because damaged sites in the genome can be converted to permanent mutations during DNA replication. The susceptibility of a particular cell type to carcinogenesis is related to its relative abilities to metabolize genotoxic carcinogens and to repair damaged DNA (81). Furthermore, susceptibility to genotoxic damage partially depends on the temporal relationship among DNA damage, DNA repair, and DNA replication (82). It...

Fish Vs Other Techniques For Cytogenetic Analysis

For the obvious reasons like high sensitivity, technical ease, applicability to a wide range of clinical specimens, and the feasibility of combining with phenotypic characterization, FISH has been welcomed in routine clinical diagnostic practices. FISH, while taking advantage of the foundation of cytopatho-logical and histopathological competency, provides an additional spectacle revealing genomic secrets of the cellular pathology. This matrimony of cell and molecular biology is thus a major...

Multiple Thermal Amplification

Ligase Chain Reaction In multithermal target amplification (specifically LCR), a high temperature is used to denature or unzip the double-stranded DNA (dsDNA) target Fig. 5. linked linear amplification. Fig. 5. linked linear amplification. (26,27). Multiple probes complementary to different regions of the target then bind to their specific targets in the annealing step. The next step in the LCR reaction takes place at the optimal temperature for the DNA polymerase so that elongation or...

Molecular Genetics In Endocrinology

Severe Igf Deficiency Pathophysiology

THE GROWTH HORMONE PATHWAY The growth hormone (GH) pathway is comprised of a series of interdependent genes whose products are required for normal growth (see Fig. 1). The GH pathway genes include ligands (GH and insulin-like growth factor 1 or IGF-1), transcription factors (PIT1 and PROP1), agonists (GH-releasing hormone or GHRH), antagonists (somatostatin), and receptors such as the GHRH receptor (GHRHR) and the GH receptor (GHR). These genes are expressed in different organs and...

C A C A T

Fig. 2. (A) Nucleotide differences between the RHCE and RHD genes. Numbers indicate nucleotide positions, and epitope-related positions are illustrated in black. Note that, for simplicity, only RHCE and RHce haplotypes are shown (RhcE and RHCe haplotypes exist). (B) Strategy for typing RHCc Ee alleles by ASPCR targeting nucleotides 48, 307, and 676. (C) RHC c genotyping by ASPCR and 2 agarose gel electrophoresis. Products of each analysis are run in sets of three lane 1 the RHC intron 2...

Amplification Refractory Mutation System

All PCRs depend on the annealing of oligonucleotide primers to specific sites in target DNA, facilitating amplification of a unique sequence. For the polymerase to extend the primers, they must be perfectly annealed to the target sequence at their 3' ends. The amplification refractory mutation system (ARMS) exploits this by utilizing a primer that is designed such that its 3' end matches, for example, one of two alternatives at a mutant nucleotide (40). ARMS is ideally suited to the detection...

Interpretation

Many areas of pathology are by their nature subjective, and IHC is no exception. What is considered positive will depend, to varying degrees, on the intensity of staining, the distribution of stain within the cell, and or the percentage of cells showing staining. Ideally, the interpretation will be based on criteria set forth in the literature, but this is not always the case, especially because the literature frequently gives divergent data. The variation in published reports can be the result...

Clinical Verification

Laboratory methods provide information for use in managing patients and addressing relevant clinical questions. The usefulness of a method depends on both the analytical performance and the clinical characteristics of the method (clinical sensitivity, specificity, and predictive value). These last factors are characteristic of the clinical application and not properties of the test. The clinical significance of a test should be defined in terms of the disease or syndrome that is the subject of...

References

Association studies between the agniotensin-converting enzyme insertion deletion polymorphism and hypertension still interesting J. Hypertens, 20 1049-1051, 2002. 2. Jones, A. and Montgomery, H. The gly389arg beta-1 adrenoceptor polymorphism and cardiovascular disease time for a rethink in the funding of genetic studies Eu . Heart J. 23 1071-1074, 2002. 3. Ridker, P. M., Hennekens, C. H., and Miletich, J. P. G20210A mutation in prothrombin gene and risk of myocardial...

Earliest Medicine

Long before there were laboratories, there were accepted practices for patient evaluation. Early health care providers (not all were physicians) attempted to determine the health status of the person under evaluation by any means possible. The diagnosis and the prescribed treatment were not always an accurate or scientifically based pronouncement. The process was motivated by a combination of altruism, vanity, greed, scientific thought, and philosophical and religious edict. This is not meant...

Applications Of Dna Sequencing In The Clinical Laboratory

Microsatellite Instability

In the area of clinical molecular genetics, the number of targets for sequencing-based mutation analysis is limited only by the number of genes in the human genome and the number of genes associated with human disease. Although some com Fig. 20. Slab gel sequencing with radiolabeling run in mutation scanning mode. The autoradiogram shows eight patient samples, a negative control (leftmost lane in each loading) and a positive control, TGC to TAC at codon 609 or C609Y rightmost lane in each...

Unsupervised Learning

Introduction Commonly used unsupervised learning techniques fall broadly into two categories (hierarchical and partitioning), of which hierarchical clustering is more widely used (90). The preference for hierarchical clustering is at least twofold after considering the following challenges of machine learning. Computational resources to solve many machine learning problems are not currently available. The chasm is broached by abandoning the search for an exact solution, accepting,...

Clinical Applications Of Flow Cytometry

Routine applications of flow cytometry in the clinical laboratory include enumeration of peripheral blood CD4+ T-cells in patients with human immunodeficiency virus (HIV) infection, enumeration of CD34+ stem cells in peripheral blood and bone marrow for use in stem cell transplantation, and immunopheno-typic characterization of acute leukemias, non-Hodgkin's lymphomas, and other lymphoproliferative disorders. In certain instances, the DNA content, or ploidy status of tumor cells, which is...

Implementation Of Laboratory Developed Tests

TEST SELECTION Similar to any other clinical test, the primary goal of molecular tests should be to provide reliable and timely results necessary for patient care. The introduction of new molecular tests has the primary goal of improving patient care. Clinical needs, as well as advances in basic and translational research, often prompt the development of new molecular tests. Several key elements are highly important during test selection. It is necessary to realize that any new test should...

Clinical Tests Formats

There are two major clinical assay formats widely used in molecular diagnostic laboratories. One of the formats are those tests developed by in vitro manufacturers and the other are assays developed by each laboratory (LDA), also known as home-brew assays. 2.1. TESTS DEVELOPED BY IN VITRO MANUFACTURERS This first category of tests is composed of kits developed by manufacturing companies to provide quality-controlled reagents for the performance of the entire molecular test for a determined...

Extraction

The first steps of any extraction process are tissue isolation, disruption, and cell lysis. Again, the specific protocol required depends on the sample. When a large portion of fresh or fixed solid tissue is available, it is important to select appropriate areas for subsequent harvest of nucleic acid. For collection of genomic DNA or RNA, tissue cannot be autolyzed or necrotic. Focal necrosis is common in many solid tumors, and sampling of these areas provides little or no intact nucleic acid....

Software For Data Analysis

A discussion of the full range of bioinformatic tools available for sequence analysis, data mining, and genomics is beyond the scope of this chapter. Comments here will be limited to software for base calling in automated sequencers. In some respects, the discssion is necessarily brief, as the algo-rithims used by manufacturers of automated sequence analyzers are proprietary. However, Green and colleagues have developed base calling software, phred, that is nonproprietary, is well described in...

Quality Control And Quality Assurance Of The Testing Process

Every molecular diagnostics laboratory should develop a comprehensive written quality assurance program. The objective of the quality assurance program is to objectively and systematically monitor and evaluate the quality and appropriateness of the test results. The quality assurance program should address every aspect of the testing process preanalytical, analytical, and post-analytical processes. The program must include written policies and documentation for the education and training of...

Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is an autosomal recessive disorder with a carrier frequency of 1 50, which affects 1 6000 to 1 10,000 live-born children. Thus, SMA is the second most common autosomal recessive lethal disease after cystic fibrosis. In this disorder, anterior horn cells degenerate, resulting in hypoto-nia, symmetrical muscle weakness, and wasting of voluntary muscles. The childhood spinal muscular atrophies are divided into three types (I, II, III) according to age of onset, rate...

Trinucleotide Repeat Disorders

Trinucleotide repeat expansions, initially described in 1991 (1), are responsible for a number of neurological diseases, including Fragile X syndrome (FRAXA), myotonic dystrophy (DM), spinal and bulbar muscular atrophy (SBMA), Huntington's disease (1HD), Friedreich ataxia (FRDA), spinocerebellar ataxias (types SCA1, SCA2, SCA3, SCA6, SCA7, SCA8, and SCA12), and dentatorubral-pallidoluysian atrophy (DRPLA) (2-5). A number of other rare forms of SCA continue to be identified in families, but...

Sample Source

There are, essentially, two types of tissue available for nucleic acid analysis fresh and preserved. The ideal source of nucleic acid is, naturally, fresh tissue. If extraction is not possible immediately, it is critical to rapidly limit the damaging action of tissue endonucleases. Prompt flash-freezing of solid tissue with liquid nitrogen preserves nucleic acids and can facilitate subsequent tissue and cell disruption. Timely freezing is especially important to extract RNA successfully. For...

Standards

Interpretation of microarray experiments is not only the task of the investigator performing the experiment but also the scientific journal peer reviewers, the readers of those journals, and other interested parties. The scale of the genomic data in addition to its complexity has challenged all involved to seek new methods of communication in order to take full advantage of its promise. An early realizations was that print media is insufficient to allow full interpretation of these data-rich...

Nucleic Acid Storage

When nucleic acid must be stored, either for archival purposes or before assay performance, the key goal is prevention of enzymatic or physical damage to the purified product. Three chief weapons are available for this endeavor chelating agents, chaotrophic agents, and refrigeration. As discussed earlier, special care is needed when the solution contains RNA or high-molecular-weight DNA. In general, DNA can be stored effectively for long periods in a Tris-EDTA buffer at 4 C. The chelation of...

Pcrbased Methods For The Detection Of Dna Methylation

BISULFITE MODIFICATION Any methylation information that is present in the original DNA is lost as soon as the DNA is amplified in vitro. Thus, in order to study methylation using PCR, the DNA must be modified in some way that can be replicated. Sodium bisulfite treatment, which deaminates cytosine to uracil, has become the method of choice for modification. The rate of deamination of 5-methylcytosine to thymine is very much slower than the conversion of cytosine to uracil (49). Bisulfite...

Microarray Study Design And Sources Of Error

We briefly mentioned measurement error in Section 3.2 with regard to fluorescence ratios however, measurement error enters into the array experiment at multiple steps (29). Consider the following experiment for potential sources of error. Liver biopsies are performed on two different mouse populations one with normal livers and the other with tumors. Messenger RNA is prepared from each of the biopsies. The RNA is labeled and then hybridized against a microarray, and the array is scanned. The...

Polymerase Chain Reaction

In a typical PCR, successive synthetic cycles are performed in which DNA polymerase copies a target DNA sequence from a template molecule in vitro (Fig. 1). The amplification products of each cycle provide new templates for the next round of amplification. Thus, the concentration of the target DNA sequence increases exponentially over the course of PCR. The typical PCR mixture contains (1) a thermostable DNA polymerase (Taq poly-merase), (2) target-specific forward and reverse...

I I I I I I Ii I I I I I I I I I I I I Ii I I I I I I I I I I I I I I I I I I

'I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I I Fig. 4. Pronto assay. A conventional PCR is carried out, followed by enzymatic degradation of unincorporated dNTPs. A 5'-labeled primer is then annealed to the PCR and DNA polymerase and a biotinylated dNTP added. The biotinylated dNTP is only added to the primer if it matches the target amplicon. The reaction mix is then added to a streptavidin-coated well and biotinylated primers are thus bound unbound primers...

Dna Sequence Variations Mutation Andor Polymorphism

As noted earlier, only approx 5 of the approx 3 billion basepairs that constitute the human genome codes for proteins. Scientists have begun to understand the significance of the sequences that comprise the remaining 95 . One interesting aspect of these noncoding sequences is the amount of interindividual variation that these sequences exhibit. In some cases, these sequence variants are the result of mutation, the random alteration of DNA. When two or more sequence variants are present in a...

Interpretation Of The Southern Blot

Once the membrane that contains the target DNA sequences has been probed with a specific labeled probe, the results of the Southern analysis can be interpreted. Positive signals on the membrane are created when a labeled nucleic acid probe binds to complementary target DNA sequences, producing a band or bands that can be visualized. If nonra-dioactive, colorimetric probing techniques were used, the bands would be visible to the naked eye. Chemiluminescent and radioactive probes must be...

Overview Of Coagulation

GENERAL CONCEPTS The components of coagulation include the blood vessels, platelets, coagulation factors and cofactors, and the fibrinolytic proteins. As a result, hemostasis is a delicate balance between surface proteins on vessels suben-dothelium, surface glycoproteins on platelets, procoagulant and anticoagulant proteins, and fibrinolytic proteins (3). The interaction among these components results in both fibrin clot formation and dissolution at the site of injury. Hereditary or...

Clinical Application Of Bioinformatics

IDENTIFICATION OF DISEASE GENES The isolation of human disease genes has important implications not only for understanding the molecular and cellular basis of the disease but also for prevention and treatment. Cloning the gene responsible for an inherited disease leads to the potential for developing molecular diagnostic tests for mutant alleles of the gene, such as the detection of mutations by PCR. A test can be developed that can identify individuals at risk for the disease and enable...

Laboratory Considerations

Cytogenetics is used to detect gross abnormalities of the human chromosomes that are large enough to be seen with aid of the light microscope. To study human chromosomes, the cells must be dividing, as the individual chromosomes are only visible at this level during mitosis. Routine analyses are performed at metaphase, although techniques to collect cells earlier, in prophase or prometaphase, can be used, as mentioned previously. With the exception of bone marrows, most samples From Molecular...

Eugene H Lewis III Myra J Lewis Jean A Amos and Gregory J Tsongalis

Cystic fibrosis (CF), a clinically heterogeneous disease, is the first genetic disease for which adult population screening has been initiated in the United States. Since the discovery of the CFTR gene in 1989, much has been learned about the pathophysiology and molecular genetics of this disorder. This review includes an overview of the genetics of CF, a discussion of pathophysiology, and clinical and anatomic pathology and concludes with a review of molecular diagnostics. Cystic fibrosis is...

Comparative Genomic Hybridization A

Genomewide screening of the gains or losses of the specific chromosomal regions came into sight with the development of comparative genomic hybridization (CGH). (for review, see refs. 38 and 47). Importantly, such screening is possible from interphase cells, where CGH is superior over M-FISH, SKY, or Q 1. How stable are the probes Can I freeze-thaw them Can I store them diluted A Generally recommended storage temperature is -20 C, at which the probes are stable. They can be repeatedly frozen...

Clinical Implications Of Cytogenetic Assessment By Fish

Since the pioneering demonstrations of the association of chromosomal abnormalities with human diseases like Down's syndrome, Turner's syndrome, CML, APL, and so forth as described earlier, several other congenital disorders and cancers have been now linked with specific chromosomal abnormalities. With this knowledge and combined by the effective cyto-genetic analysis tools, chromosomal banding and, among the sensitive molecular assays, FISH, genomic disease management is seeing the light of...

Pcr Components

NUCLEIC ACID TEMPLATES FOR PCR Polymerase chain reaction amplifies specific sequences from DNA templates (either genomic DNA or cDNA derived from RNA) that can be prepared from various sample sources (Fig. 2). This DNA could be genomic DNA isolated directly from experimental or patient material or it could be cDNA that has been synthesized from DNA or RNA templates by polymerase or reverse transcriptase enzymes. Various sources of biological material can be utilized for the preparation of...

Drug Metabolism And Distribution

As described earlier, the field of pharmacogenetics began with examinations of polymorphisms in drug-metabolizing enzymes. Although the field has expanded well beyond drug-metabolizing enzymes, this aspect of pharmacogenetics continues to be the most thoroughly developed. In large part this is because these traits are primarily monogenic (polymorphisms in one gene are responsible for the observed phenotype) and highly penetrant (likely to influence phenotype). The metabolism of any given drug...

Increasing Pcr Specificity And Sensitivity

Several approaches are available that can improve upon the specificity and sensitivity of various PCR applications. Some of these approaches utilize specialized reagents, while others employ modified PCR cycling parameters or variations of oligonucleotide primer design. 5.1. IMPROVED TAQ POLYMERASE PREPARATIONS Several specialized PCR reagents have emerged over the last decade that function to improve PCR specificity. These include special preparations of Taq polymerase. Taq polymerase has...

Dna Microarray Platforms

Microsatellite Instability Probes

In the following subsections, we will develop that DNA microarray model introduced in section 2 by detailing the two most commonly used DNA microarray platforms. The basic principles for the cDNA array remains true to that first described by Schena and Brown. The authors helped ensure wide distribution of the technology by making available protocols for building a robot spotter to produce DNA chips, a scanner to analyze them, and the software to run the experiment (17). With these protocols, a...

Hetero Duplex Analysis

Heteroduplex Analysis

Detection of p53 sequence variants by sscp analysis. A 183-bp fragment of exon 8 of the p53 gene was amplified from DNA extracted from four different breast tumor cell lines and human placenta (wild-type control). 33P-labeled dATP was included in the PCR to label the products that were separated by electrophoresis through a 0.5X MDE gel at room temperature. Lane 1 Wild-type control lane 2, MDA-MB-231 cells lane 3, MDA-MB-468 cells lane 4, Bt-474 cells lane 5 SkBr3 cells. The cell lines...

Microsatellite Instability Rtpcr

ISOLATION OF RNA One of the most important factors in generating high-quality full-length cDNA is the purity and integrity of the total RNA used as starting material. Chaotropic agents such as guanidinium chloride and guani-dinium isothiocyanate are capable of dissolving cellular structures and proteins, causing nucleoproteins to dissociate rapidly from nucleic acids and inactivating RNAse enzymes. Several procedures using chaotropic and reducing agents for the isolation of RNA from cells...

Nonenzymatic Signal Amplification

Hpv Testing With Well Plates

Hybrid Capture The hybrid capture kits from Digene Inc. (Gaithersburg, MD) can be used to detect HPV, CT GC, and HSV. Full-length RNA probes are used for the detection of the HPVs and to determine whether they have a Fig. 6. Nonenzymatic signal amplification. Fig. 6. Nonenzymatic signal amplification. high or low risk for oncogenic potential. Full-length probes negate the potential for false negatives because SNPs do not inhibit hybridization. The hybrid consists of the viral DNA with...

Molecular Tests In The Clinical Coagulation Laboratory

GENERAL ASPECTS The applications of molecular diagnostics in coagulation testing are confined mainly to patients with thrombosis, an area for which laboratory testing has increased steadily over the past years. The availability of molecular diagnostics for hereditary thrombophilic disorders depends on the underlying molecular defect. Based on their unique molecular abnormalities, the FVL mutation, Prothrombin G20210A mutation, and the MTHFR mutation all lend themselves to relatively...

Quality Assurance Quality Assessment

A quality assurance (QA) program that is established, implemented, and maintained by a laboratory can help ensure high-quality results are provided in clinically relevant turnaround times. Its major goal is to minimize laboratory errors by continual assessment and subsequent improvement of the services provided. Such a program is to cover all steps of the testing operation, including the preanalytical, analytical, and postana-lytical processes, which must be continuously monitored and assessed....

Capillary Electrophoresis

Microsatellite Electrophoresis

Capillary electrophoresis (CE) is an extremely powerful instrumental technique that has introduced automation into the clinical molecular laboratory. The method, originally described by Jorgenson and colleagues at the University of North Carolina in 1983 (7), utilizes electrophoresis in the thin, fused silica capillary columns that had been recently developed for use in capillary gas chromatography. Electrophoresis in free solution (without porous matrices) in open tubes was not new, having...

General Principles Of Flow Cytometry

Flow cytometry is a technique in which single cells in a fluid suspension are analyzed with respect to their intrinsic light-scattering properties and are simultaneously evaluated for one or more extrinsic properties (i.e., the presence of specific molecules) using fluorescent probes. The fluorescent probe might bind directly to the targeted molecule (e.g., propidium iodide in DNA content analysis) or a fluorescent dye might be coupled to an antibody probe, to enable detection of a specific...

Maternal Contamination And The Sensitivity Of Genotyping

Microsatellite Instability

Maternal contamination of umbilical cord blood, chorionic villus, and amniocentesis samples is a well-documented problem that presents itself to laboratories conducting prenatal genetic testing (79-82). Because analysis by molecular diagnostic methodologies does not distinguish maternal from fetal DNA, maternal contamination presents a risk for misidentifica-tion of the fetal genotype and possible misdiagnosis. The reality of this problem is further emphasized when one considers that the cell...

Acquired Chromosomal Abnormalities

The human genome is inherently unstable with mutations occurring at a low background rate. Most of these changes are lethal and do not survive or they are innocuous and therefore of Type of abnormality deletion Critical region 22q11.21-11.23 Thymus hypoplasia or aplasia with deficit in cellular immunity Diminished number of T-cells Hypoparathyroidism Cardiac anomalies Subtle dysmorphic features Velo-cardio-facial syndrome Type of abnormality deletion Critical region 22q11.2 Long face Smooth...

Electrophoresis Of Restrictiondigested

Electrophoresed Genomic Dna Bands

DNA Nucleic acids are negatively charged at a neutral pH, which allows their migration through an electric field 8 . Agarose is a highly porous polysaccharide that acts as a sieve, allowing the fragments of DNA to be separated according to length. Under low-voltage conditions, the electrical resistance of all components remains constant and the linear DNA fragments move through the agarose gel at a velocity proportional to the voltage applied. The driving force for nucleic acid migration in the...

Alcidine Orange Cell Staining

Checking Rna Integrity Agarose Gel

RNA was isolated from cultured rat liver epithelial cells by the acidic phenol extraction method. Two micrograms of RNA per sample were run on an integrity gel for 2 h at 90 V. Lane A contains an RNA molecular size standard. Lanes B and C contain intact RNA rRNA bands are clearly visible , whereas the samples in lanes D and E are degraded. The RNA in lane D was prepared in a small volume of tap water instead of RNAase-treated water and the RNA in lane E came in...

Constitutional Chromosomal Abnormalities

Microsatellite Instability

Chromosomal abnormalities occur quite commonly. Most are not compatible with life and are aborted spontaneously. Over 50 of early SABs have an abnormal karyotype 21 . The attrition rate of fetuses with chromosomal abnormality decreases as pregnancy progresses, and about 5 of stillbirths 21 and a smaller percentage of term deliveries are chromosomally abnormal. Chromosomal abnormality is a significant cause of major birth defects. Abnormalities of the sex chromosomes are generally better...

Robert Bagnell Jr

Advances in our understanding of disease mechanisms have resulted in the need for single-cell analysis. Analytical technologies have become available to accommodate such interrogations. Typically, molecular diagnostic assays begin with a nucleic acid extraction procedure during which tissue architecture and cellular morphology is lost. Laser capture microdissection LCM is a technology that enables scientists to examine the processes of individual cells. Whether one is investigating a cell's...

Dna Sequencing

Amino Acid Microsatellite Instability

DNA sequencing is considered the gold standard for mutation detection. However, before proceeding with a description of methods and technologies, it is important to point out that sequencing is not perfect, and it certainly is not magic. Like any analytical method, one can encounter false positives and false negatives. However, when properly done and properly interpreted, sequencing a DNA fragment will almost always reveal a high percentage, near 100 , of the sequence variations present....

The Molecular Endocrinology Of Diabetes Mellitus

Diabetes mellitus is a metabolic syndrome characterized by hyperglycemia resulting from variable defects in insulin secretion and action. Traditionally, diabetes mellitus has been divided into four categories Type 1 diabetes, characterized by absolute insulin deficiency and requirement of insulin therapy to sustain life Type 2 diabetes, characterized by variable defects in insulin secretion and action, without an absolute need for insulin therapy although insulin might be necessary for adequate...

Fluorescence In Situ Hybridization

Fluorescence in situ hybridization essentially indicates the fluorescent reporting of ISH. A sketch of routine FISH protocols for embedded tissue sections and cell preparations shown in Table 2 highlights its origin in the common ISH platform. FISH is commonly identified with its most popular application in cytogenetics, in contrast to the general notion of ISH as a RNA- detecting technique. In recent years, the growing commercialization of FISH technology has guided its way into the disease...

Preparation Of Labeled Nucleic Acid Probes

Once nucleic acids have been affixed to a membrane, specific sequences can be detected by hybridization with a labeled, denatured, single-stranded probe that binds to homologous RNA or DNA. These probes might be composed of either RNA or DNA, and labeling methods might be radioactive or nonra-dioactive. 11.1. NICK TRANSLATION The method of nick translation relies on Escherichia coli DNA polymerase I a polymerizing enzyme that also possesses a 5' 3' exonuclease activity that degrades...

Fundamentals Of Electrophoresis

Microsatellite Instability

Electrophoresis refers to the migration of charged molecules through a liquid or gel medium under the influence of an electric field. Zone electrophoresis, or the migration of macromol-ecules through a porous support medium, or gel, is almost universally used in molecular biology laboratories today. Electrophoresis all discussion in this chapter will involve zone electrophoresis but will be referred to as electrophoresis for brevity is a powerful separation tool, being able to detect the...

The Rh Blood Group

The Rhesus Rh blood group system contains antigens produced from two distinct genes, RHD and RHCE, that are tandemly localized on chromosome 1 and are thought to have arisen through duplication of a single ancestral gene 15,16 . The RH genes are greater than 95 homologous at the nucleotide sequence level, both consist of 10 exons spanning over 75 kb, and both encode peptides of 417-amino-acid residues, with a predicted molecular mass of 30-35 kDa 17-19 . Approximately 15 of Caucasians are...

Analytical Verification

Before a new or improved LDT is introduced into the laboratory menu, careful evaluation of performance characteristics of the assay under laboratory conditions needs to be done. In addition to evaluating these characteristics, undertaking analytical verification can provide useful information with regard to issues of practicality that will have to be addressed before introducing the LDA. It is important to point out that the performance of analytical validation programs has been challenging...

Duchenne Muscular Dystrophy

Duchenne muscular dystrophy DMD is a neuromuscular disorder that also involves gene deletion and duplication. DMD is a devastating, progressive, muscle-wasting disorder that is usually not diagnosed before the age of 3, but often results in wheelchair confinement by age 12 and death in the early twenties. Manifestations of this disease include pseudohypertrophy of muscles, joint contractures, scoliosis, respiratory compromise, cardiomyopathy, and markedly increased serum creatine kinase CK...

Indications For Chromosomal Evaluation

Human Metaphases

AMNIOTIC FLUID The reasons for karyotypic evaluation vary by sample type. Amniotic fluids are the primary sample for prenatal evaluation. Genetic amniocenteses are ideally performed at about 16 wk of gestation and are generally offered to women when their risk of having a chromosomally abnormal fetus is greater than the inherent risk of the amnio-centesis procedure itself. Early amniocentesis can be performed at 10-12 wk. The most common reason for performing a genetic amniocentesis is...

Genotyping Platelet And Neutrophil Antigen Systems

Neonatal alloimmune thrombocytopenic purpura NATP , the platelet counterpart of HDN, occurs with a frequency of approx 1 in 1100 live births 70 . Infants affected with NATP are at risk of bleeding and 10-30 of cases develop intracranial hemorrhage 50 in utero , which can result in brain damage and lasting neurological sequelae. There are currently 25 different platelet-specific alloantigens and antigens shared with other tissues that have been implicated in causing NATP Table 1 . Maternal...

Platelet Glycoproteins

Platelets work in concert with coagulation factors to maintain hemostasis. In ACS, platelet activation plays a major role in the arterial thrombosis of coronary arteries. Whereas a fibrin clot dominates in patients with totally occluded ST-segment AMI, platelet clots are more responsible for the partial occlusions observed in non-ST-segment AMI and unstable angina 16 . There are several glycoprotein GP receptor complexes found on the platelet surface that facilitate the binding functions of...

Multiple Endocrine Neoplasia Type

Multiple Endocrine Neoplasia

Multiple endocrine neoplasia type 1 MEN1 is an autosomal dominant trait with marked intrafamilial variability, characterized by multiple tumors or hyperplasia of endocrine glands see OMIM 131100 . MEN1 is defined by the presence of two of the following three endocrine tumors parathyroid adenoma or hyperplasia, entero-pancreatic endocrine tumors, and pituitary adenomas. Other possible manifestations include carcinoid tumors, adrenal adenomas, and lipomas. The MEN1 gene was identified through...

General Procedures For

SPECIMEN FIXATION With regard to in situ molecular detection techniques, the most basic influencing factor is specimen fixation, which serves to preserve morphology and retain the necessary target nucleotide sequences. The common fixatives in use are formalin, Carnoy's fixative methanol acetic acid , Bouin's solution picric acid , and ethanol. Of these, buffered formalin seems to best preserve DNA and mRNA in a tissue section 21 . Formalin brings about target protein and nucleic acid...

Target Vs Signal Amplification

Transcription Mediate Amplification

Nucleic acids can be detected using either target or signal amplification methods. Briefly, target amplification enzymati-cally increases the number of target molecules. In short, at the end of the day, there are more molecules of the targeted nucleic acid. In contrast, signal amplification does not increase the target but uses highly sensitive reporter molecules or probes to detect the target. For example, in signal amplification, the same number of molecules exists at the end of the day, but...

Markers For Heart Failure

ANGIOTENSIN-CONVERTING ENZYME The angiotensin-converting enzyme ACE is a critical enzyme in the rennin-angiotensin-aldosterone RAA axis. Under the action of renin, angiotensinogen is first degraded to the decapeptide angiotensinI and then to the octapeptide angiotensinII by ACE. Angiotensin II is further degraded into a heptapeptide angiotensin III by angiotensinase. When there are periods of blood and fluid loss, the RAA axis is essential in maintaining blood pressure by stimulating...

Microsatellite Acrylamide

Micro Satellite Instability

Detection of mutations and sequence variants by heteroduplex analysis of the CFTR gene. Exon 10 of the CFTR gene was amplified by PCR for heteroduplex analysis. The gel is 1.5 mm thick and 40 cm long 1X MDE Cambrex Bio Science Walkersville, Inc. containing 15 urea. The gel was run for 21,000 V-h in 0.6X Tris-borate-EDTA buffer and stained with ethidium bromide. Lanes 1 and 13 are size markers 100 bp ladder , lane 2 is a MM homozygote at postion 470, lane 3 is a VV homozygote at position 470, lane 4 is a heterozygote for the M470V polymorphism, lane 5 is a homozygote for the AF508 mutation, lane 6 is a AI507 wild-type heterozygote, lane 7 is a compound heterozygote for AF508 and I506V, lane 8 is compound heterozygote for AF508 and F508C, lane 9 is a compound heterozygote for AF508 and Q493X, lane 10 is an I506V wild-type heterozygote, lane 11 is a F508C wild-type heterozygote, and lane 12 is a AF508 wild-type heterozygote. of heteroduplexes vs homoduplexes in a set of...

Direct Vs Indirect Molecular Diagnostic Tests

Genetic testing, through interrogation of DNA, RNA, chromosomes, or proteins, can provide critical information for the detection of heritable disease genotypes for a number of different applications Table 3 15 . Many procedures can be used in either a direct or indirect mutation analysis. Most types Applications of Molecular Genetic Testing Diagnostic testing Testing for a gene mutation in symptomatic individuals as a diagnostic aid. Newborn screening Testing is used to screen populations to...