Abstract
Molecular genetic methods have become an organic part of everyday clinical practice. In the past, molecular diagnostic tests were carried out for genetic diagnosis of a particular monogenic disease. In these situations the tests itself were used for identification of one particular genetic alteration (e.g., point mutation or deletion) of the gene of interest. Later, parallel with the development of the technology, the focus has shifted by allowing investigating at once targeted gene panels and even the whole exome/genome behind a suspected genetic disorder. Historically for these purposes, array-based methods (oligonucleotide arrays) and then next-generation sequencing-based methods have been used. High-throughput methods have been fundamentally transforming the everyday, routine genetic diagnostics, but older molecular techniques still have a role in clinical genetics. Here, we summarize the most important molecular genetic methods and shed light to the advantages and disadvantages of their application in routine diagnostics. We mainly focus on methods used for detection of germline alterations.
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Abbreviations
- aCGH:
-
Array comparative genomic hybridization
- ARMS:
-
Amplification refractory mutation system
- AS-PCR:
-
Allele-specific PCR
- cDNA:
-
Complementary DNA
- CGH:
-
Comparative genomic hybridization
- CNV:
-
Copy number variations
- cT:
-
Cycle threshold
- DDGE:
-
denaturing gradient gel electrophoresis
- ddNTP:
-
Dideoxyribonucleotide triphosphate
- DNA:
-
Deoxyribonucleic acid
- dNTP:
-
Deoxyribonucleotide triphosphate (adenine, cytosine, guanine, and thymine triphosphate: dATP, dCTP, dGTP, and dTTP, respectively)
- dPCR:
-
Digital polymerase chain reaction
- dsDNA:
-
Double-stranded DNA
- EDTA:
-
Ethylenediaminetetraacetic acid
- FRET:
-
Fluorescence resonance energy transfer
- HA:
-
Heteroduplex analysis
- HRM:
-
High-resolution melting analysis
- IVD:
-
In vitro diagnosis
- MLPA:
-
Multiplex ligation-dependent probe amplification
- NGS:
-
Next-generation sequencing
- OD:
-
Optical density
- PASA:
-
PCR amplification of specific alleles
- PCR:
-
Polymerase chain reaction
- qPCR:
-
Quantitative polymerase chain reaction
- RFLP:
-
Restriction fragment length polymorphism
- RT-qPCR:
-
Reverse transcription quantitative polymerase chain reaction
- SNP:
-
Single-nucleotide polymorphism
- SNV:
-
Single-nucleotide variants
- SSCP:
-
Single-strand conformation polymorphism (SSCP)
- STR:
-
Small tandem repeat
- UPD:
-
Uniparental disomy
- VAF:
-
Variant allele frequency
- VUS:
-
Variant of uncertain significance
- WES:
-
Whole-exome sequencing
- WGS:
-
Whole-genome sequencing
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Acknowledgments
This work has been supported by the National Excellence Program to Attila Patócs and ÚNKP-18-4-SE-8 New National Excellence Program of The Ministry of Human Capacities to Henriett Butz. Henriett Butz is a recipient of Bolyai Research Fellowship of Hungarian Academy of Sciences.
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Butz, H., Patócs, A. (2019). Brief Summary of the Most Important Molecular Genetic Methods (PCR, qPCR, Microarray, Next-Generation Sequencing, etc.). In: Igaz, P., Patócs, A. (eds) Genetics of Endocrine Diseases and Syndromes. Experientia Supplementum, vol 111. Springer, Cham. https://doi.org/10.1007/978-3-030-25905-1_4
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