MANIPULATING GENOMES
(a) The principles of DNA sequencing and the development of new DNA sequencing techniques.
● To include the rapid advancements of the techniques used in sequencing, which have
increased the speed of sequencing and allowed whole genome sequencing e.g. high-
throughput sequencing.
The Sanger method, or the capillary method, involves sorting DNA fragments of increasing
length and marking each one with a fluorescent terminator base, identifying what the next base
in the sequence is. The DNA for sequencing is mixed with a primer, DNA polymerase, and an
excess of nucleotides, and modified fluorescent terminator bases. The mixture is placed in a
thermal simulator that performs the temperature changes required for the polymerase chain
reaction. At 60oC, DNA polymerase synthesizes a DNA strand complementary to the single-
stranded DNA by adding free nucleotides with complementary base pairing. Each time a
terminator base is incorporated, the the oxidised base terminates the synthesis. After many
cycles, all the possible lengths of DNA chain will be produced with the reaction stopping at every
base. The DNA fragments are separated by gel electrophoresis in minute capillary tubes and the
fluorescent markers on the terminator bases are used to identify the final base on each fragment
with photosensors identifying the light signature created when the fragments are scanned with a
laser. The length of the fragment is then used to order the bases in the sequence. This data is
compiled by a computer to comparing all the fragments and finding areas that overlap.
High throughput sequencing methods have since been developed. Pyrosequencing uses
sequencing by synthesis, not by chain termination. It involves synthesising a single strand of
DNA, complementary to the strand to be sequenced, one base at a time, whilst detecting, by light
emission, which base is added.
A long length of DNA to be sequenced is mechanically cut using a nebuliser into fragments 300-
800 base pairs and degraded into a single-stranded ssDNA, which are immobilised and act as a
template. A sequencing primer is added and the DNA is then incubated with DNA polymerase,
ATP sulfurylase, luciferase, apyrase, and the substrates adenosine 5’ phosphosulfate (APS), and
luciferin. One activated nucleotide—ATP, TTP, CTP, GTP—is incorporated into the
complementary strand of DNA at a time and as this happens two of the phosphoryl groups are
released as PPi, which in the presence of APS, ATP sulfurylase converts to ATP, and in the
presence of ATP, luciferase converts luciferin to oxyluciferin, which generates visible light,
detected by photosensors. One million sequences are read any one time by software.
(b) (i) How gene sequencing has allowed for genome-wide comparisons between individuals and
between species.
● With reference to bioinformatics and computational biology and how these fields are
contributing to biological research into genotype–phenotype relationships, epidemiology
and searching for evolutionary relationships.
Bioinformatics is the development of software and computing tools needed to organise and
analyse raw biological data; computational biology then uses this data to build models of
biological systems and analyse the data, allowing genome-wide comparisons to be made. These
entire genomes to be easily compared and analysed.
By comparing entire genome sequences, patterns of methylation and location of single
nucleotide polymorphisms, between groups of individuals with different phenotypic
characteristics, the functions of some genes can be determined, and the effect of different alleles
in contributing to disease. This helps to identify genetic predispositions, mutations causing
