DNA Sequencing

What is Sanger sequencing?

First described by Frederick Sanger and colleagues in 1977, Sanger sequencing was the most widely used DNA sequencing method for over 25 years. The basic building block of DNA is the nucleotide. The nucleotide consists of one of four nitrogenous bases, Adenine (A), Cytosine (C), Guanine (G) or Thiamine (T), a deoxyribose sugar and a phosphate group. In a human cell, a strand of DNA is built by special enzymes called DNA polymerases that bind the phosphate group from one nucleotide to a carbon from the sugar of another nucleotide. DNA is double-stranded and forms base pairs, A with T and C with G. DNA polymerase “reads” one strand, called the template strand, and uses it to build the complementary strand.

DNA can also be synthesized in a test tube by placing the four different types of nucleotides into a tube with the DNA to be sequenced and adding the polymerase enzyme. DNA polymerase will continue to bind nucleotides together according to the template strand until there are no free-floating nucleotides left in the solution.

The Sanger sequencing method is based on the discovery that certain alterations to the phosphate group on a nucleotide will cause the polymerase to stop adding bases and fall off the template. The modified phosphate groups on the different nucleotides (A, T, C, and G) can be labeled for detection (previously radioactive labels were used, now fluorescent labels are used).

Once labeled, scientists can determine the order in which the bases are added (they can read the DNA “sentence”) because they could see where the fluorescent labels were added. In one tube, all the As fluoresced and in another tube, all the Cs fluoresced. By separating the DNA fragments by length and putting the results right next to each other, scientists can easily read which base came next, thereby deducing the DNA sequence.

Sanger sequencing is a very accurate method of DNA sequencing. However, because it is slow and expensive, it is not a feasible method for sequencing large amounts of DNA in a timely manner. Most clinical DNA sequencing today is accomplished by next generation sequencing techniques.

What is Next-Generation Sequencing?

Next-generation sequencing (NGS), sometimes called massively parallel DNA sequencing, produces thousands to millions of DNA sequencing reactions at the same time. A single gene or a large number of genes can be sequenced quickly and in a cost-effective manner, making NGS an effective high-throughput method for clinical DNA testing. In NGS, a patient’s DNA is sheared into many small fragments, and each of these fragments is sequenced many times over. The millions of sequencing reads are then mapped to the reference sequence, which serves as a standard, or a control, because the sequence is already known. Variations in a patient’s DNA are detected when enough of the sequencing reads from the patient’s DNA differs from the reference sequence.

NGS is a very accurate way to detect single nucleotide substitutions (patient has a C at a particular position, while the reference sequence has a G, for example). However, NGS is not as accurate at detecting small insertions and deletions, or correctly reading homopolymers. A homopolymer is a DNA sequence in which one base is repeated several times in a row; for example TTTTTTTTTT. Because of this limitation, most laboratories that test patient samples do a second test to confirm that the results are accurate.

NGS is also not able to reliably detect large insertions of DNA (>20 base pairs), large deletions of DNA (>50 base pairs), or copy number variants (CNVs) and other structural variants. No single test platform is perfect and it can be necessary to combine platforms for the best results.

In addition to offering NGS for many genes, Claritas also offers separate tests to detect missing or extra pieces of genes. These are known as deletion/duplication assays and are only performed for genes in which large deletions and/or duplications are known to be associated with disease. In addition, the Claritas Genomics offers the ClariView Array, a chromosome microarray test that assesses for copy number variants across the genome.