The development of techniques for rapid sequencing of DNA was one of the most significant advances in molecular genetics. Currently, it costs far less to sequence a stretch of DNA than to sequence and characterize the amino acid peptide that the DNA produces.
Although other mutation screening techniques exist, sequencing of DNA is the surest and most direct. Sequencing of complementary DNA (cDNA) derived from mRNA provides a quick look at the reading frames (exons) of the gene, whereas sequencing of genomic DNA is more time-consuming because of the presence of introns between the exons. The intron–exon boundaries must be known and multiple PCR assays set up in order to screen not only the exons and their splice junction sites but also upstream and downstream regions that may be important for gene activation and regulation.
DNA sequencing techniques currently in use include the enzymatic (or Sanger sequencing) method, which can be automated (Fig 5-4), and next-generation sequencing (NGS), also known as massively parallel sequencing. NGS offers the ability to sequence the entire genome of an individual. Some NGS methods use as probes allele-specific oligonucleotides that are constructed to employ hybridization to recognize a specific DNA sequence in order to detect a specific point mutation (Fig 5-5).
Early methods of mutation detection included Sanger sequencing using radioactive and later fluorescent probes; the single-stranded conformational polymorphism (SSCP) technique; denaturing gradient gel electrophoresis (DGGE); and the use of RFLPs.
Whole-exome sequencing will identify many potential mutations; however, identification of true disease-causing mutations will require considerable bioinformatic information.
Zhang J, Chiodini R, Badr A, Zhang G. The impact of next-generation sequencing on genomics. J Genet Genomics. 2011;38(3):95–109.
Excerpted from BCSC 2020-2021 series: Section 2 - Fundamentals and Principles of Ophthalmology. For more information and to purchase the entire series, please visit https://www.aao.org/bcsc.