# Copy Number Variation: Methods and Clinical Applications
## 2 Methods of CNV Detection
### Methods of Molecular Biology  
Molecular-biology methods such as Southern blot hybridization offer higher resolution than cytogenetics [21]. The principle of Southern blotting relies upon fragmentation of DNA with a restriction endonuclease and separating fragments by gel electrophoresis. The fragments are transferred to a membrane and hybridized to appropriate probes. Copy number changes are visible as differential hybridization intensities or as altered mobility of the fragments. Although for many years, Southern blotting was the standard method for the detection of deletions or amplifications in the range of 5-500 kb [22], it is a laborious, time-consuming method that requires large amounts of high-quality DNA [23]. A tremendous improvement in the screening of CNVs came with the introduction of microarray-based methods, specifically in conjunction with comparative genomic hybridization, where DNA samples extracted from the tested and reference cells are color-bridized to an array of fixed oligonucleotide probes instead of metaphase chromosomes [24]. The aCGH provides genome-wide coverage at a much higher resolution of 10-25 kb [25] or even >500 bp if high-density arrays are used [26]. Despite some limitations in resolution and accuracy, this made aCGH a standard in CNV detection [27]. PCR-based methods may either come in the form of conventional two- or three-primer based protocols or multiplexed assays. CNVs may be detected by PCR-based protocols through the change of: (i) migrational properties during agarose gel or other types of electrophoresis [28]; (ii) amplification cycles required to achieve a relative threshold fluorescent intensity in real-time quantitative PCR (qPCR) assays [29; 30]; (iii) relative fluorescent signal intensities in capillary electrophoresis when using quantitative-fluorescent PCR (QF-PCR) [31] or multiplex ligation-dependent probe amplification (MLPA) techniques [32]; (iv) denaturation properties reflected in melting temperatures or melting curve shapes during conventional or high-resolution melting analysis [33]. All of these methods are more or less convenient for the targeted detection of a limited number of CNVs in a relatively wide range of length from tens of bp up to Mb and even whole chromosomes at low cost and fast turnaround time [34]. However, each of these methods also has its own advantages and limitations. A more recently introduced alternative to the traditional qPCR in CNV detection is the droplet digital PCR (ddPCR). In this method, template DNA is diluted and partitioned into thousands of nano-scale droplets of uniform volume, allowing for absolute quantification of target copy numbers without the need for a standard assay, making results easier to interpret and less error-prone than regular qPCR [35]. Another PCR-based method is multiplex amplifiable probe hybridization (MAPH) using oligonucleotide probes that hybridize to a specific region in the genome. Hybridized probes are amplified and the amount of each amplification product is proportional to the copy number of the corresponding sequence [36]. MAPH enables the sensitive detection of CNVs as small as 150 bp [37]. Even more sensitive and easier to use is MLPA developed to determine the copy number of multiple genomic DNA sequences (up to 60 probes) in a single reaction with resolution from a single nucleotide difference. The probes hybridized to the sample DNA are ligated and amplified, resulting in fragments of a unique length which can be separated and quantified by capillary electrophoresis [32]. Therefore, MLPA is a cost-effective method that can be performed with equipment present in most molecular biology laboratories. Although the first generation of DNA sequencing (IGS) technologies, specifically Sanger sequencing, was generally considered to be the gold standard in DNA diagnostics, CNVs represent specific challenges not easily dealt with when using this method.