Genetic and genomic investigations
Angus Clarke, Alex Murray, Julian Sampson in Harper's Practical Genetic Counselling, 2019
Multiplex ligation-dependent probe amplification (MLPA) is an application of the polymerase chain reaction (PCR) in which it is not the patient's DNA that is amplified by PCR but, instead, carefully designed probe sequences. Two DNA probes are designed to recognise and anneal to immediately adjacent sequences in a gene of interest; once in place, they are ligated together by a DNA ligase. The two probes carry an additional sequence, not complementary to human DNA sequences, including PCR primer binding sites and additional ‘stuffer sequences’ designed so that the PCR product is of a very specific size. The PCR reaction of the probe sequence, not human sequence, can then proceed, yielding a quantity of product that relates directly to the number of copies of the target DNA in the patient's genome (Figure 5.1).
Genetic Basis of Neuromuscular Disorders
Maher Kurdi in Neuromuscular Pathology Made Easy, 2021
On the other hand, multiplex ligation-dependent probe amplification (MLPA) is another technique widely used for the detection of copy number change, deletions, or duplications in single genes. For example, MLPA could be useful for a screening of patients who could benefit from Exondys 51 and have an exon 51 skipping in the DMD gene. The MLPA assay is a modified form of multiplex polymerase chain reaction where all targeted exons are hybridized into two specially modified oligonucleotide probes that will bind targets adjacent to each other. This binding is sequence specific and highly sensitive to mismatches. Following successful ligation of the two probes, the amplification of that is detected on DNA analyzers. Because MLPA uses a dedicated reference for every gene, the MLPA targets can be quantified and a deletion or duplication is detected.
Bannayan–Riley–Ruvalcaba Syndrome
Dongyou Liu in Handbook of Tumor Syndromes, 2020
The diagnosis of BRRS is made clinically, despite the absence of specific diagnostic criteria. BRRS patients harboring a PTEN mutation are grouped in the PHTS assortment. Mutations and/or deletions within PTEN are identified by genetic testing. Detection of such alterations confirms the PHTS diagnosis and permits predictive testing and prenatal diagnosis among patients’ relatives [14]. Several methods are presently employed to detect PTEN deletions. These include multiplex ligation-dependent probe amplification (MLPA) (the preferred method), Southern blotting, monochromosomal hybrid analysis, real-time polymerase chain reaction (PCR), and semiquantitative multiplex PCR [14]. In order to optimize yield, the best order of PTEN testing should be first sequencing complete PTEN coding exons 1–9 and flanking intronic regions. The second step, if no pathogenic modification is found, would be analysis for deletion/duplication [30]. A more accurate and comprehensive assessment of chromosomal abnormalities can be achieved combining chromosomal microarray analysis and conventional cytogenetics in order to identify anomalies in chromosomes [31].
Histological Evaluation of Products of Conception, Who Benefits from It?
Published in Fetal and Pediatric Pathology, 2023
Haleh Soltanghoraee, Arash Mohazzab, Azadeh Soltani, Soheila Ansaripour, Maryam Tavakoli, Maryam Rafati, Amir Hassan Zarnani, Saeed Reza Ghaffari
Samples were received in normal saline without any fixative, in a cool box with ice. They were examined macroscopically and the size of the gestational sac and embryo, if present, was recorded. Then 2–3 samples of chorionic villi, membranes, umbilical cord or embryo (if included) were taken and transferred to separate microtubes, and were stored in −20 °C for probable use in genetic tests. There were also 2–3 samples submitted from the sac, embryo and decidua which were prepared for histology. All macroscopic evaluations were performed by an experienced perinatal pathologist. After fixation in 10% buffered formalin, the tissue was processed in an automated tissue processor and embedded in paraffin, with 4-5 micron sections stained with H&E. All samples were assessed microscopically by the same pathologist who evaluated them macroscopically at first. Immunohistochemistry staining for confirmation of histiocytic origin of intervilli cells in CIUE group was performed using CD 68 antibodies (mouse monoclonal antibodies, clone KP1, EDTA pretreatment buffer, incubation, DAB staining, Diagnostic Biosystem, Netherlands). Some of the samples were referred to the genetics lab for MLPA (Multiplex Ligation-dependent Probe Amplification) per their practitioner’s order.
Advances in genetic testing and optimization of clinical management in children and adults with epilepsy
Published in Expert Review of Neurotherapeutics, 2020
Marcello Scala, Amedeo Bianchi, Francesca Bisulli, Antonietta Coppola, Maurizio Elia, Marina Trivisano, Dario Pruna, Tommaso Pippucci, Laura Canafoglia, Simona Lattanzi, Silvana Franceschetti, Carlo Nobile, Antonio Gambardella, Roberto Michelucci, Federico Zara, Pasquale Striano
As a second step, karyotype may be helpful to identify possible chromosome rearrangements that are not detectable by array CGH, such as translocations. FISH is only indicated in selected cases to search for known deletions or duplications in specific syndromes (e.g., 22q11.2 deletion in suspected Di George syndrome) or to better define chromosome abnormalities identified with other techniques (e.g., the complex rearrangements in the duplication/inversion 15q11 or isodicentric 15 chromosome syndrome). Eventually, multiplex ligation-dependent probe amplification (MLPA) allows to detect deletions and duplications of several exonic sequences and can be used for the screening of entire genes in the same experimental session. Furthermore, MLPA plays a pivotal role in the identification of intragenic deletions in cases where Sanger sequencing and array CGH result negative (e.g., epilepsy due to SCN1A or CDKL5 intragenic deletions).
Association between AKT2 gene polymorphism and polycystic ovary syndrome: a case-control study
Published in Gynecological Endocrinology, 2021
Jie Li, Sien Mo, Yan Sun, Hua Huang, Shujia Wang
The whole blood was collected from all participants after acquiring informed consent. Genomic DNA was extracted from peripheral blood leukocytes using a DNA isolation kit following the manufacturer’s instructions (Sangon Biotech, Shanghai, China). The genotyping was mainly based on the SNPscan method (Genesky Biotechnologies Inc., Suzhou, China). The details were presented in other studies (http://biotech.geneskies.com/en/index.php/Index/fuwuer/id/29) [17–18]. Briefly, this method was a multi-gene mutation screening technology, which ameliorated the previous multiplex ligation-dependent probe amplification technology to some extent. During the process of this method, four different fluorescent dyes were applied. In addition, the ligations were lengthened, which could help to detect more SNPs simultaneously. In order to ensure the veracity of the genotyping, quality control assessment was also performed during the genotyping process. The laboratory personnel were blinded to the case-control status of the samples, with a blank control used for all PCR amplifications. Finally, 10% of samples from patients and controls were randomly selected to evaluate the quality of genotyping, which showed 100% concordance.
Related Knowledge Centers
- Amplicon
- Brca1
- Denaturation
- DNA
- Fluorescence
- Gel Electrophoresis
- Multiplex Polymerase Chain Reaction
- Nucleic Acid Hybridization
- Polymerase Chain Reaction
- Primer