each with a different dye attached (Section 8.5.4, Figure 8.11). The four sequencing reactions are carried
out together in one reaction and subjected to capillary electrophoresis in free solution. As each length of
fluorescently labelled oligonucleotide emerges from the capillary column, it is detected by a fluorescence
detector, the particular colour in each case corresponding to one of the four dideoxynucleotides. A com-
puter analyses the data and produces a series of fluorescent signals that correspond to the read sequence.
Such machines are capable of generating sequence reads of 500–1000 residues.
Sequencing machines have proved to be essential in large-scale DNA sequencing of genomes. For example,
the DNA sequences of the yeast Saccharomyces cerevisiae, the fruitfly Drosophila melanogasterand Homo
sapienshave been determined in this way. In genome sequencing, it is usual for the sequence to be deter-
mined three to ten times from different clones or fragments, including from both strands of the DNA, to
obtain higher accuracy. Powerful computer programmes are then able to determine overlaps between frag-
ments, and align the sequences against genome maps (Section 6.5.2). The positions of genes, introns and alter-
native splicing patterns can be predicted and genomes compared between different organisms to obtain
knowledge of the RNA transcripts as well encoded proteins.
5.1.3 RNA Sequencing by Reverse Transcription
It is possible to carry out base-specific chemical treatments or digestions by nuclease enzymes to determine an
RNA sequence directly. However, a more common procedure is to use a reverse transcriptaseenzyme to
make a cDNA copy of the single-stranded RNA. The reaction is initiated by use of an oligodeoxyribonucleotide
primer from a known part of the sequence. This cDNA can then be amplified by the polymerase chain
reaction(PCR) (Section 5.2.2) and sequence determined by standard DNA sequencing. The method only
gives information regarding the base sequence and not regarding RNA modifications.
5.2 Gene Cloning
Cloningis the technique of growing large quantities of genetically identical cells or organisms that are
derived from a single ancestor (clones). Gene cloningis an extension of this whereby a particular gene, group
of genes or a fragment of DNA is selected from a mixed population (often a complete genome) and ampli-
fied to a huge extent. This can be carried out by insertion of the chosen DNA into a vector DNAand intro-
duction of the hybrid (recombinant DNA) into cells by transformation(transfection). The cells containing
the recombinant DNA are propagated and each cell in a colony contains an exact copy (or copies) of the gene
‘cloned’ in the vector. Cloning and recombinant DNA technology are well documented in established text-
books and manuals.6,7
Nowadays, a separate and complementary approach that uses PCR can achieve the same objective in a
fraction of the time, of amplification of DNA segments without involving living cells.
5.2.1 Classical Cloning
5.2.1.1 Vectors. Several classes of vector exist into which a foreign DNA can be inserted and ampli-
fied. The major classes are plasmids, bacteriophageand cosmids and bacterial or yeast artificial chromo-
somes(YACs). Prokaryotic plasmids are almost always circular double-stranded DNAs that contain antibiotic
resistance genes as markers and a variety of restriction sites that can be used for insertion of the foreign DNA.
A large number of plasmids are available for use in E. coli. The most useful bacteriophage is, which has
been engineered in many ways to accept inserts of many different sizes and types, up to approximately
20,000 base pairs. Because the transformation frequency is high, screening is easy. Cosmids are large plas-
mids that contain the packaging site for bacteriophage DNA. Therefore they can either be packaged into
phage particles or they can be replicated as plasmids. Since the amount of DNA that can be packaged in a
phage particle is 50,000 base pairs, the potential size of cosmid inserts is very large. Cosmids have been
used in chromosome walking(see later this section), but they are somewhat more difficult to manipulate
than is . Artificial chromosomes are vectors containing the constituents of natural yeast chromosomes,
170 Chapter 5