Introduction
DNA sequence variations are commonly found in humans, in fact, any pair of unrelated human genomes are estimated to vary by one nucleotide for every 300 nucleotides. Although these variations may have little to no effect on the phenotype due to being within introns or silent mutations that do not alter the translated amino acid sequence, there are many hereditary diseases that are caused by variations in single gene sequences. Moreover, studies of cancer at a molecular level have highlighted the significance of driver mutations in the growth and metastasis of tumors. Parallel to this, differences witnessed in pathogen DNA resulting from sequence variations have led to varying impacts on human health, with antibiotic resistance emerging as a significant global healthcare challenge.
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Within genomics and disease research, there are several technologies that have been developed to detect and quantify sequence variations. These technologies are primarily categorized into three methodologies and their combinations: polymerase chain reaction (PCR), hybridization, and next-generation sequencing (NGS). Each method brings with it unique technical and operational advantages and disadvantages.
Fig. 1 Detection of sequence variants using allele-specific PCR primers (Khodakov D., et al. 2016).
Polymerase Chain Reaction
PCR is a method that amplifies a template DNA molecule using synthetic DNA primers, a DNA polymerase, and dNTPs. This method is the most widely employed for the detection of DNA sequences. Quantitative PCR specifically, is considered to be more accurate than either microarrays or NGS for DNA and RNA quantitation. However, PCR's downfalls are its inability to perform highly multiplexed assays due to primer dimer formation causing a high frequency of false positive results or false negatives.
A technique employed to detect sequence variations using PCR entails using oligonucleotide reagents to amplify the variant of interest more efficiently than the corresponding wildtype DNA sequence. However, for somatic mutation analysis in biopsy samples where the variant may be at a low as 5% allele frequency, ARMS primers fail to consistently provide adequate sequence discrimination.
Alternative approaches to allele-specific PCR introduce the use of two-segment primers or a variant of blocker PCR. The former offers a longer 5' region and a shorter 3' region to improved specificity. The latter relies on the use of blocker oligonucleotides which suppress wildtype amplification. This implies the variant sequence does not need to be known ahead of time and it offers compounded specificity through multiple cycles of PCR. As a result, it is uniquely suitable for the detection of the presence or absence of pathogen genes, rather than detailed sequence variations.
Hybridization
Hybridization, a phenomenon where synthetic DNA bonds with a biological DNA target, forms the foundation of many modern DNA analysis and diagnostic techniques. Although extremely useful in many contexts, a major limitation of the process is that it doesn't offer sequence amplification. This shortfall necessitates pairing the process with a signal amplification technology or a sensitive readout instrument.
Microarrays are one way that the multiplex readout problem in hybridization is solved. The process involves attaching diverse DNA probes onto a surface at varied positions. The resulting fluorescent spots are indicative of the identities and quantities of the DNA targets detected. Microarrays are arguably one of the most viable methods of capturing highly quantitative information regarding nucleic acid concentration. They have been used in many diagnostics that have received clearance or approval from the FDA, such as those for tracking the recurrence of breast cancer, understanding the genomic polymorphisms that influence therapeutic responses to antidepressants and antiepileptics, and evaluating intellectual disabilities, developmental delays, and congenital anomalies based on the analysis of chromosomal mutations. However, these have their limitations, with significant annotation bias observed across different genes, transcripts, and microarray platforms.
Another prominent technological advancement in hybridization is the fluorescent barcode method, which is another means of achieving high-level multiplexed readout. This technique uses the relative intensities of different fluorophores to identify sequence identity. In principle, this method allows up to 900 barcode variations. An FDA-approved diagnostic panel for predicting the recurrence of breast cancer was built based on fluorescent barcodes.
In situ hybridization (ISH), another technique driven by hybridization, presents not only sequence and concentration data for genes of interest, but can also show their positioning within the tissue. This makes ISH especially suited for analyzing copy number variations in heterogeneous cell or tissue samples.
Next-Generation Sequencing
NGS gives the opportunity for the massively multiplexed sequence analysis of DNA and RNA. Unlike traditional Sanger sequencing, NGS permits the analysis of heterogeneous samples, providing sequence information for millions of randomly selected nucleic acid molecules in a sample at once.
However, no chemistry is flawless, and NGS platforms have an intrinsic error rate. Sequencing errors can complicate the calling of variants, especially low frequency variants. Recent innovations in molecular barcoding have significantly decreased the NGS error rates, however, increasing sequencing depth is necessary in doing so.
Ampliseq, droplet-based enrichment, and ligation-based enrichment are several methods that are used to allow NGS analysis of low-volume samples. Ampliseq uses a proprietary enzymatic reaction and yields major advantages such as low DNA requirements and fast protocol completion. Droplet-based Enrichment involves droplets each containing different primers which prevent the issues caused by primer-primer reactions. Subsequently, new primer pairs can be added to the process without causing disruptions to the existing panel. Finally, ligation-based enrichment uses a pair of primers that bind to the same strand of the template. Non-specific binding and/or extension of either the first primer or the second primer to other regions of the genome does not result in amplicons with both adapter sequences.
In conclusion, the genome is a complex and intricate landscape, and understanding the variations within it is fundamental for improvements in genome diagnostics and therapeutics. By furthering the development, precision, and usability of PCR, Hybridization and NGS technologies, we are continually expanding our ability to detect and quantify sequence variations in DNA and thereby furthering our understanding of genetics and disease.
Reference
- Khodakov D., et al. Diagnostics based on nucleic acid sequence variant profiling: PCR, hybridization, and NGS approaches. Advanced Drug Delivery Reviews. 2016, 105: 3-19.
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