Comparison of different methods for telomere length measurement in whole blood and blood cell subsets: Recommendations for telomere length measurement in hematological diseases.

Different methods of telomere length measurement are used to identify patients with telomeropathies. In our lab, we established four different methods for telomere length measurement, terminal restriction fragment (TRF) analysis by Southern blot analysis, quantitative PCR (qPCR), quantitative telomere/centromere fluorescence in situ hybridization (T/C-FISH) and fluorescence in situ hybridization combined with flow cytometry (FlowFISH). The methods each have distinct properties and apart from this-according to our experience and data-may have an impact on the individual result. In this study, we therefore compared and validated these methods measuring 154 healthy individuals of different age groups (newborn-81 years). A linear decline was found for every method (Southern blotting 64 bp per year; qPCR 31 bp per year; T/C-FISH 36 bp per year; FlowFISH 50 bp per year). With the equation of the regression line the values of each method (T/S ratio, T/C value, RTL value) can be expressed in absolute kb. All methods showed acceptable accuracy. The analysis indicated good agreement between all methods, with the best agreement between T/C-FISH and FlowFISH. Here, FlowFISH was the most precise, accurate, and reproducible method compared to the other methods. Based on our data, we emphasize the influence of expertise and experience that is required to produce robust and reliable telomere length analyses. Furthermore, we want to provide the scientific community working in diagnostics and research with data-funded advice on how to choose the appropriate method to safely discriminate between natural variability and pathological telomere shortening in individual cases.

Blame it on Southern, but it's a western blot.

Edwin M. Southern is a professor emeritus at the University of Oxford. He is perhaps best known for development of the "Southern blot" (Dr. Southern was at the University of Edinburgh when he wrote his landmark paper). The Southern blot provided a scientific breakthrough by allowing scientists to detect a particular DNA sequence without first purifying it from the rest of the genome; the basic method involves the transfer of the DNA to a membrane, followed by detection with a specific probe. Although few people perform Southern blots as originally carried out by Southern, due in part to the more recent technique of the polymerase chain reaction, the basic concept continues to play an important role in molecular biology.

The gene cluster instability (GCI) assay for recombination.

A newly developed method for quantitatively detecting genomic restructuring in cultured human cell lines as the result of recombination is presented: the "gene cluster instability" (GCI) assay. The assay is physiological in that it detects spontaneous restructuring without the need for exogenous recombination-initiating treatments such as DNA damage. As an assay for genotoxicity, the GCI assay is complementary to well-established sister chromatid exchange (SCE) methods. Analysis of the U-2 OS osteosarcoma cell line is presented as an illustration of the method.

Validation of a reference gene (BdFIM) for quantifying transgene copy numbers in Brachypodium distachyon by real-time PCR.

Brachypodium distachyon has been proposed as a new model system for gramineous plants with a sequenced genome and an efficient transformation system. Many transgenic B. distachyon plants have been generated in recent years. To develop a reliable fast method for detecting transgenic B. distachyon and quantifying its transgene copy numbers, a species-specific reference gene is of great priority to be validated both in qualitative PCR and quantitative real-time PCR detection. In this study, we first proved that the BdFIM (B. distachyon fimbrin-like protein) gene is a suitable reference gene in qualitative PCR and quantitative real-time PCR for B. distachyon. Fourteen different B. distachyon varieties were tested by both qualitative and quantitative PCRs, and identical amplification products of BdFIM were obtained with all of them, while no amplification products were observed with samples from 14 other plant species, suggesting that BdFIM gene was specific to B. distachyon. The results of Southern blot analysis revealed that the BdFIM gene was low copy number in seven tested B. distachyon varieties. In conclusion, the BdFIM gene can be used as a reference gene, since it had species specificity, low heterogeneity, and low copy number among the tested B. distachyon varieties. Furthermore, the copy number of inserted sequences from transgenic B. distachyon obtained by real-time PCR methods and Southern blot confirmed that the BdFIM gene was an applicable reference gene in B. distachyon.

Genomic Southern blot analysis.

This chapter describes a detailed protocol for genomic Southern blot analysis which can be used to detect transgene or endogenous gene sequences in cereal genomes. The protocol follows a standard approach that has been shown to generate high-quality results: size fractionation of genomic DNA; capillary transfer to a nylon membrane; hybridization with a digoxigenin-labelled probe; and detection using a chemiluminescent-based system. High sensitivity and limited background are key to successful Southern blots. The critical steps in this protocol are complete digestion of the right quantity of DNA, careful handling of the membrane to avoid unnecessary background, and optimization of probe concentration and temperatures during the hybridization step. Detailed instructions on how to successfully master these techniques are provided.

Detection of clonal IGH gene rearrangements: summary of molecular oncology surveys of the College of American Pathologists.

CONTEXT: The diagnosis of B-cell lymphoid malignancy can frequently be substantiated by detecting clonal immunoglobulin heavy chain (IGH) gene rearrangements, which is typically done by polymerase chain reaction (PCR) amplification and/or Southern blot analysis. OBJECTIVE: To characterize current laboratory practice for the assessment of IGH rearrangements and to identify opportunities for improvement. DESIGN: The data from the Molecular Oncology Proficiency Survey distributed to participating laboratories by the Molecular Pathology Committee of the College of American Pathologists from 1998 through 2003 were analyzed. RESULTS: Thirty-nine proficiency survey specimens (29 positive and 10 negative for clonal IGH rearrangements) were distributed. For Southern blot analysis, 944 results were reported, with a successful response rate of 95%. For PCR detection, 2349 results were reported, with a successful response rate of 72%. A higher rate of successful responses by PCR was achieved using framework 3 primers in combination with other frameworks (82%) compared with framework 3 primers only (76%) and when fresh/frozen (72%) compared with paraffin-embedded (65%) tissues were analyzed. CONCLUSIONS: The performance of the participating laboratories was very good, by both Southern blot and PCR analysis. As expected, Southern blot analysis consistently detects a higher proportion of IGH rearrangements than PCR analysis. Further improvement and standardization of the IGH PCR assay is important if it is to replace Southern blot analysis as the standard method. Participation in this survey is a valuable tool for assessing laboratory performance and it directs our attention to areas where we may improve laboratory practice.

False-positive diagnosis of a single, large-scale mitochondrial DNA deletion by Southern blot analysis: the role of neutral polymorphisms.

Single, large-scale deletions of mitochondrial DNA (mtDNA) are a common finding in the molecular investigation of patients with suspected mitochondrial disorders and are typically detected by Southern blot analysis of muscle DNA that has been linearized by a single cutter enzyme (BamHI or PvuII). We describe our investigations of a 47-year-old woman with exercise intolerance, myalgia, and ptosis who underwent a muscle biopsy for a suspected mitochondrial genetic abnormality. Southern blot analysis after digestion of muscle DNA with BamHI revealed the apparent presence of two mtDNA species, indicative of a heteroplasmic deletion of 2.0-2.5 kb in length involving approximately 50% of all molecules. Contrary to this observation, longrange polymerase chain reaction (PCR) amplified only wild-type mtDNA. Sequence analysis revealed that the patient harbored two previously recognized control region polymorphisms, a homoplasmic 16390G>A variant that introduces a new BamHI site and a heteroplasmic 16390G>A change that abolishes this site, thus explaining the initial false-positive testing for a heteroplasmic mtDNA deletion. Our findings highlight the potential problems associated with the diagnosis of mitochondrial genetic disease and emphasize the need to confirm positive cases of mtDNA deletions using more than one enzyme or an independent method such as long-range PCR amplification.

Early loss of deleted in colorectal carcinoma gene transcript detected in a group of benign colon adenomas.

We examined the expression of the putative tumor suppressor gene deleted in colorectal carcinoma (DCC) in human colon adenoma tissues and cell lines. One allele of DCC is deleted in 70% of human colon carcinomas, and DCC expression is undetectable in 90% of colon carcinoma cell lines. One DCC allele is also deleted in 50% of human colon adenomas, but results from protein expression studies have differed as to whether complete loss of DCC expression could occur in colon adenomas, or instead correlates with progression of colon adenoma to carcinoma. To further examine the timing of DCC expression loss in colon adenomas, we assayed DCC transcript levels in adenoma cell lines and tissues. We measured DCC expression by a sensitive assay using Southern blot detection of the RT-PCR-amplified DCC transcript. DCC expression was negligible or greatly reduced in 4 of 14 colon adenomas, including 2 of 2 adenoma cell lines and 2 of 12 adenoma tissue samples. These data are the first evidence that expression of DCC transcript can be silenced in colon adenoma cell lines and tissues. These data indicate that loss of DCC expression occurs in some colon adenomas, but is insufficient to drive the adenoma to carcinoma progression.

Detection of genetically modified organisms in foods.

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. In this article, protein- and DNA-based methods employing western blots, enzyme-linked immunosorbant assay, lateral flow strips, Southern blots, qualitative-, quantitative-, real-time- and limiting dilution-PCR methods, are discussed. Where information on modified gene sequences is not available, new approaches, such as near-infrared spectrometry, might tackle the problem of detection of non-approved genetically modified (GM) foods. The efficiency of screening, identification and confirmation strategies should be examined with respect to false-positive rates, disappearance of marker genes, increased use of specific regulator sequences and the increasing number of GM foods.

Incidence of MLL rearrangement in acute myeloid leukemia, and a CALM-AF10 fusion in M4 type acute myeloblastic leukemia.

To determine the incidence of the mixed lineage leukemia (MLL) gene rearrangements in acute myeloid leukemia (AML) without cytogenetically-detected 11q23 abnormalities, we screened 64 cases of AML at diagnosis for MLL rearrangement by FISH. Three cases (4.7%) had a MLL rearrangement detected; one was shown to have a cryptic t(11;22)(q23;q11) and another to have a t(9;11)(p21-22;q23) which had been missed by the conventional cytogenetic study. No 11q23 structural abnormality was visible in the third case. Twenty-six of the 64 cases were further studied by Southern blotting and DNA hybridization, and four of these cases (15%) were found to have MLL rearrangement: in three of these, FISH had not detected any abnormality. FISH was also used to confirm MLL involvement in eight cases of AML that had a cytogenetic abnormality at 11q23; in one of these, Southern blot did not show a rearrangement. The survival of patients with MLL abnormalities identified by cytogenetics, FISH and/or DNA analysis was significantly worse than that of patients without MLL abnormalities (event-free survival p = 0.016) although two patients with a t(9;11)(p21-22;q23) were long-term survivors, consistent with this particular translocation having a better prognosis. One further case with a cytogenetic abnormality close to 11q23 was studied; it was found to have a t(10;11)(p13;q21), and the breakpoints were shown by FISH to involve the Clathrin Assembly Lymphoid Myeloid (CALM) gene at 11q21 and the AF10 gene at 10p13. Our data confirm the value of combining cytogenetic, FISH and molecular analyses to define the incidence and precise nature of MLL and 11q23 abnormalities in AML.

Cytogenetic and molecular monitoring of residual disease in chronic myeloid leukaemia.

For patients with chronic myeloid leukaemia, methods for monitoring response to treatment have changed considerably in recent years. In the 1980s, the principal approach was repeated examination of bone marrow metaphases for the presence of the Ph chromosome in patients treated by interferon-alpha (IFN-alpha) or allogeneic stem cell transplantation. The use of fluorescence in situ hybridisation (FISH) techniques to detect the BCR-ABL fusion gene in Ph-positive leukaemia cells increased the sensitivity of cytogenetic studies to some degree. In the last 10 years, the reverse-transcriptase polymerase chain reaction (RT-PCR) has proved extremely valuable for assessing and monitoring minimal residual disease in patients who achieve Ph negativity after treatment with IFN-alpha or with the new Abl tyrosine kinase inhibitor imatinib mesylate or after allogeneic stem cell transplantation (SCT). Results are consistent with the notion that the majority of long-term survivors after allogeneic SCT are probably 'cured'; for other patients monitored serially in complete cytogenetic remission, rising numbers of BCR-ABL transcripts detected by RT-PCR can indicate the need for further therapy.

[External quality assessment of the genetic testings for hematopoietic tumor, CML].

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.

Isolation of genomic DNA from feathers.

The use of feathers in veterinary clinical practice simplifies the sampling of avian genomic DNA, especially when blood extraction is difficult because of the age or the size of the bird. A rapid and accurate protocol was used to isolate high-quality genomic DNA from feathers. The technique includes a lysis step of the feather quill, which differs in temperature and time of incubation depending on the feather size. Purification of genomic DNA is performed with phenol: chloroform: isoamyl alcohol extraction and ethanol precipitation. This protocol consistently provided significant amounts of high-quality genomic DNA from more than 800 birds belonging to 120 different species. Genomic DNA isolated with this method was used for Southern blotting and also in several polymerase chain reaction systems devoted to sex determination and paternity testing.

Accuracy of prenatal diagnosis for haemoglobin disorders in the UK: 25 years' experience.

We have reviewed the accuracy of prenatal diagnosis for the thalassaemias and sickle cell disorders performed for UK residents since the service began in 1974. Prenatal diagnosis has been performed in 3254 pregnancies: 517 by fetal blood analysis, 681 by Southern blotting and 2056 by polymerase chain reaction (PCR) methods, the majority using the amplification refractory mutation system (ARMS). The number of homozygotes diagnosed was 808 (24.8%). Twenty-five diagnostic errors have been recorded, ten arising from non-laboratory errors (0.31%) and 15 due to technical problems associated with the diagnostic techniques. The latter group consisted of eight misdiagnoses by globin chain synthesis (1.55%), five by Southern blot analysis (0.73%) and two by PCR methods (0. 10%). The data show that the accuracy of prenatal diagnosis has improved with each development of diagnostic technique, and confirms that prenatal diagnosis of beta-thalassaemia and sickle cell disorders by ARMS-PCR is very accurate and reliable. The overall error rate for prenatal diagnosis by PCR methods in the UK is now 0. 41%.

Overcoming the errors of in-house PCR used in the clinical laboratory for the diagnosis of extrapulmonary tuberculosis.

Our experiences from 1993 to 1997 in the development and use of IS6110 base PCR for the diagnosis of extrapulmonary tuberculosis in a routine clinical setting revealed that error-correcting processes can improve existing diagnostic methodology. The reamplification method initially used had a sensitivity of 90.91% and a specificity of 93.75%. The concern was focused on the false positive results of this method caused by product-carryover contamination. This method was changed to single round PCR with carryover prevention by uracil DNA glycosylase (UDG), resulting in a 100% specificity but only 63% sensitivity. Dot blot hybridization was added after the single round PCR, increasing the sensitivity to 87.50%. However, false positivity resulted from the nonspecific dot blot hybridization signal, reducing the specificity to 89.47%. The hybridization of PCR was changed to a Southern blot with a new oligonucleotide probe giving the sensitivity of 85.71% and raising the specificity to 99.52%. We conclude that the PCR protocol for routine clinical use should include UDG for carryover prevention and hybridization with specific probes to optimize diagnostic sensitivity and specificity in extrapulmonary tuberculosis testing.

Sensitive method for measuring telomere lengths by quantifying telomeric DNA content of whole cells.

Recently, a new method for measuring telomere lengths based on telomere DNA content was developed. The method, which is based on the ratio of telomere to centromere DNA content (TC ratio), is highly sensitive, allowing the analysis of small quantities of DNA. However, the method required the isolation of DNA, which can be difficult or impossible for small numbers of cells. Here, we suggest an improvement of this method that can directly estimate telomere lengths from whole cells. We optimized the method for whole cells and purified DNA and found that accurate TC ratios can be obtained from as little as 9 ng of DNA or 800 whole cells. There was no statistically significant difference between the ratios obtained with purified DNA or with whole cells, indicating that the isolation of DNA is not necessary for small samples.

Routine diagnosis of large granular lymphocytic leukaemia by Southern blot and polymerase chain reaction analysis of clonal T cell receptor gene rearrangement.

AIMS: To compare the polymerase chain reaction (PCR) assay with standard Southern blot (SB) hybridisation for the detection of clonal T cell receptor (TCR) gene rearrangements in large granular lymphocyte (LGL) proliferations; to evaluate the reliability and practicality of the methods for routine diagnostic use; and to determine the sensitivity of the PCR method. METHODS: Blood lymphocytes were isolated from 12 patients with persistent CD3+CD8+ lymphocytosis with LGL morphology. Clonal rearrangements of the TCR gene were demonstrated by SB hybridisation with a TCR beta constant probe, and by PCR amplification of portions of the TCR beta and TCR gamma genes. RESULTS: Monoclonal TCR beta gene rearrangements were detected in eight patients (67%) by PCR analysis and five patients (42%) by SB hybridisation. PCR analysis also showed that seven patients (58%) had monoclonal TCR gamma gene rearrangements. All cases which had TCR beta clonal rearrangements shown by SB hybridisation were similarly identified by PCR. Sensitivity tests suggested that the TCR beta PCR technique was capable of detecting clonality in as little as 50 pg of DNA. The TCR beta primers could detect one clonal cell in approximately 200 or more normal cells (< 0.5%), a sensitivity level that at least doubles that of the SB hybridisation technique. CONCLUSIONS: The use of PCR technology proved to be superior to SB hybridisation for the routine investigation of suspected cases of LGL leukaemia. Nine patients (75%) in this study were found to have TCR beta and/or TCR gamma monoclonal gene rearrangements. This approach is ideal for distinguishing between reactive and clonal LGL proliferation in a routine diagnostic laboratory.