17.1 DNA Sequencing
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17.1.1
Extraction of Nucleic Acids
The following steps are typical of what is required:
1. Cell separation from the medium in which they are grown by filtration or cen-
trifugation;
2. Cell lysis (i.e., disruption of the cell membranes, mechanically or with detergent,
and enzymes) and elimination of cell debris;
3. Isolation of the nucleic acids by selective adsorption followed by washing and
elution. 2
17.1.2
The Polymerase Chain Reaction
If the amount of DNA is very small, it can be multiply copied (“amplified”) by the
polymerase chain reaction (PCR) before further analysis. The following steps are
involved:
1. Denature (separate) the two strands at 95 Superscript degrees◦C (i.e., melting).
2. Lower the temperature to 60Superscript degrees◦C and add primer (i.e., short synthetic chains of DNA
that bind at the beginning, the so-called 3 prime3, end, of the sequence to be amplified).
3. Add DNA polymerase (usually extracted from the thermophilic microbe Thermus
aquaticus and hence called Taq polymerase) and deoxyribonucleose triphosphates
(dNTPs; i.e., an adequate supply of monomers); the polymerase synthesizes the
complementary strand starting from the primer.
4. Stop DNA synthesis (e.g., by adding an auxiliary primer complementary to the
end of the section of the template to be copied); go to Step 1.
The concentration of single strands doubles on each cycle up to about 20 repetitions,
after which it declines. There is of course no proofreading. Miniature bioMEMS
(lab-on-a-chip) devices are now available for PCR, which operate with only a few
nanolitres of solution, and enable much faster operation. 3
17.1.3
Sequencing
The classical technique is that devised by Sanger. One starts with many single-
stranded copies of the unknown sequence, to which a known short marker sequence
has been joined at one end. An oligonucleotide primer complementary to the marker
is added, together with DNA polymerase and nucleotides. A small proportion of the
2 This procedure may yield a preparation containing RNA as well as DNA, but RNA binds prefer-
entially to boronate and thus can be separated from DNA.
3 Note the emerging rival technology of loop-mediated isothermal amplification (LAMP) (Li et al.
2017).