Cell lines are a widely used pre-clinical models for biomedical research. However, the access to cell lines from a non-confident source results in uncontrollable cell lines of uncertain quality, the main consequence of which is the lack of reproducibility between the research results. Compliance with Good Cell Culture Practice (GCCP) is essential to have quality-controlled material independently of its nature or downstream application. Quality controls of cell lines include the tools necessary to monitor their authentication, stability, functionality, and contamination absence by using different molecular biology techniques. Many of these quality controls used to test different attributes of cell lines are based on DNA, which are presented here.
The introduction of molecular cytogenetic techniques, based on FISH, revolutionized the field of cytogenetics  by allowing the identification of complex and cryptic chromosomal alterations, without prior knowledge of chromosomal loci involved. FISH allows the study of chromosome exchanges and gene rearrangements, copy number alterations, amplification, and deletions at the single-cell level, in a complementary way to conventional cytogenetics.
FISH is based on fluorescently labelled DNA probes that hybridize to unique DNA sequences along the chromosomes, on either metaphase preparations or interphase cells. Probes and target DNAs are denatured using high-temperature incubation in a formamide or salt solution. The probe is applied in excess, so the kinetics ensure that the probe anneals to the target DNA. Probe detection is accomplished by ultraviolet-light excitement of a fluorochrome, such as fluorescein-5-thiocynate (FITC) or tetramethyl rhodamine isothiocyanate (TRITC), which is directly attached to the DNA probe, or by incubation of a hapten labelled probe with a fluorescent conjugate. The majority of probes used for laboratory purposes are commercially available .
There are wide applications of FISH, mainly in cancer research and molecular diagnosis, but also as quality control for DNA stability detection of cell lines. However, issues related with long experimental times, expensive reagents and the requirement of trained technicians must be addressed in order to increase FISH applications in cytogenetic analysis. In this sense, the protocol recently evolved towards on chip detection of chromosome alterations through microsystems for FISH analysis, reaching automation of the assay performance, reduction in probe volume, as well as reduction in assay time .
Another of the FISH technique limitations is that a small number of genes can be simultaneously quantified. Thus, to increase the throughput of this informative technique, a fluorescent barcode system for the unique labelling of dozens of genes and simultaneous hybridization, and an automated image analysis algorithm, were devised. The reliability of this multiplex approach has been demonstrated on normal human lymphocytes, metaphase spreads of transformed cell lines, and cultured circulating tumour cells. It also opens the door to the development of gene panels for more comprehensive analysis of copy number changes, including the study of heterogeneity, and of high-throughput clinical assays that can provide rapid quantification of gene copy numbers in samples with limited cellularity, such as circulating tumour cells .
On the other hand, although FISH was originally used for chromosome analysis, it is currently being replaced by aCGH or next generation sequencing (NGS). For the diagnosis of single gene defects, PCR has become highly developed and is being used. Moreover, SNP arrays for karyomapping also were recently introduced .
Copy Number Variations (CNVs), defined as genomic intervals that deviate from the normal diploid state (deletions or duplications), were collectively detected in an estimated 4.8 to 9.5% of the human genome . This includes both population-specific and individual-specific variation. An average genome contains 3 to 7 rare variant CNVs. Between 5 and 10% of individuals have CNVs larger than 500 kb in size, and 1–2% of the population carry CNVs greater than 1 Mb in size . Genome sequencing studies have shown that most bases that vary among genomes reside in CNVs of at least 1 kilobase (Kb). Population-based surveys have identified thousands of CNVs, most of which, due to limited resolution, are larger than 5 kb . However, CNVs can range from sub-microscopic events to complete chromosomal aneuploidies.
The size of a CNV and its gene density are strongly anti-correlated with its frequency. Benign CNVs are often small, intergenic, or encompass genes that can tolerate a change in copy number, whereas pathogenic CNVs (those larger than 250 kb) are significantly enriched for genes involved in development and genes with constrained evolutionary patterns of duplication and loss, or strongly associated with morbid consequences such as developmental disorders and cancer. Therefore, their functional impact has been demonstrated across the full range of biology, from cellular phenotypes, such as gene expression, to all classes of human disease with an underlying genetic basis: sporadic, Mendelian, complex and infectious . Detecting CNVs within and between populations is essential to better understand the plasticity of genome and to elucidate its possible contribution to disease or phenotypic traits .
Professional guidelines have been developed for the interpretation and reporting of clinically relevant CNVs, and consider factors such as size, genomic content, comparison with internal and external databases for population frequency information, and whether the CNV is inherited or de novo .
Diverse technologies, including aCGH and SNP microarrays, and more recently, whole genome sequencing and whole exome sequencing, have enabled robust genome-wide unbiased detection of CNVs in affected individuals and in reportedly healthy. The aCGH obtains better results than the G-banded karyotype analysis through analysis of the entire genome at a much higher resolution (1 kb in size). It was first introduced to the clinical diagnostic field with arrays that contained bacterial artificial chromosome (BAC) clones corresponding to known clinically relevant microdeletion genomic intervals. Genome advancements led to replacement of BAC clones by oligonucleotide sequences in the aCGH technology or by SNP probes.
The aCGH and SNP arrays enable genome-wide detection of CNVs in a high resolution, and therefore microarray has been recognized as the prioritized test for the pathologies detection and genetic alterations resulting from modifications in the DNA sequence. The detection of CNVs through the SNP array analysis, enables the identification of stretches of homozygosity and thereby the possible identification of recessive disease genes, mosaic aneuploidy, or uniparental disomy (UPD) if contained within a single chromosome. The patient-parent trio analysis is used to screen for the presence of any form of UPD in the patient, and can determine the parental origin of a de novo copy number variation .
However, limitations of chromosomal microarrays, both aCGH and SNP arrays, include the inability to identify copy-neutral rearrangements such as balanced rearrangements, inversions, or Robertsonian translocations. Moreover, they do not provide information regarding location of a gained copy in the genome (insertions) nor of its orientation (directly repeated or inverted) . Alternatively, evolution of aCGH technology is important, highlighting exonic SNP arrays developed to detect small intragenic copy number changes, as well as large DNA fragments for the region of heterozygosity .
On the other hand, sequencing of breakpoint junctions allows to elucidate the upstream mechanisms leading to genomic instability and resultant structural variation, whereas studies of the association between CNVs and specific diseases or susceptibility to morbid traits have enhanced understanding of the downstream effects .
Furthermore, a recent technology that may fit between aCGH and NGS is next-generation mapping of long (from 100 bp to mega-base pairs) fluorescently labelled DNA molecules imaged on nanochannel arrays, which can identify CNVs or structural abnormalities with high resolution and sensitivity .
Consistently, the outcome of a genotype analysis may be used as a quality control by ruling out cell lines cross-contamination. Moreover, it is a tool that can be used for the detection of singular fragments of the genome and that serves to identify a specific genome which would make it usable for the identification of cell lines with specific alterations. Accordingly, a simple approach compatible with routine use in the laboratory was developed for the accurate determination of cell line identity throughout the course of long-term research use by genotyping 34 SNPs . Therefore, these higher resolution techniques are a very useful tool in the detection of incorrectly identified cell lines.