A)
B)
G
A
A
C
T
C
G
A
T A
Sugar−
phosphate
backbone
A T
C G
5´
3´
3´
5´
G C
G C
A T
C
T
T
G
A
G
C
T
G C
A T
GC
A T
T A
A T
C G
T A
C G
C G
3´
5´
5´
3´
Hydrogen
bond
Figure 1.1 A) DNA double helix. Each sequence is linked together by a sugar-phosphate backbone,
and complementary sequences are held together by hydrogen bonds. 30 and 50 refer to the orientation
of the DNA: one end of a sequence has an unreacted 50 phosphate group, and the other end has an
unreacted 30 hydroxyl group. B) Denatured (single-stranded) DNA showing the two complementary
sequences. DNA becomes denatured following the application of heat or certain chemicals
DNA
Transcription
rRNA
mRNA
(ribosomal) (messenger)
tRNA
(transfer)
Ribosome
Translation
Protein
Figure 1.2 DNA codes for RNA via transcription, and RNA codes for proteins via translation
Individual 1
Individual 2
Locus 1
Locus 2
Locus 3
Locus 1
Locus 2
Locus 3
Allele A
Allele A
Allele A
Allele B
Allele B
Allele A
Allele A
Allele B
Allele A
Allele B
Allele C
Allele B
Figure 1.3 Diagrammatic representation of part of a chromosome, showing which alleles are
present at three loci. Individual 1 is homozygous at loci 1 and 3 (AA in both cases), and heterozygous
at locus 2 (AB). Individual 2 is homozygous at locus 1 (BB ), and heterozygous at locus 2 (BC ) and
locus 3 (AB )
NTS
ETS
ITS1
18S
5.8S
ITS2
28S
Figure 1.4 Diagram showing the arrangement of the nuclear ribosomal DNA gene family as it occurs
in animals. The regions coding for the 5.8S, 18S, and 28S subunits of rRNA are shown by bars; NTS ¼
non-transcribed spacer, ETS ¼ external transcribed spacer, ITS ¼ internal transcribed spacers 1 and 2.
The entire array is repeated many times
3´
5´
Replication fork
3´5´
3´
5´
3´
5´
3´ 5´
3´5´
Figure 1.5 During DNA replication, nucleotides are added one at a time to the strand that grows in a
50 to 30 direction. In eukaryotes, replication is bi-directional and can be initiated at multiple sites by a
primer (a short segment of DNA)
Locus 1
Locus 2
Locus 3
Locus 1
Locus 2
Locus 3
Allele A
Allele B
Allele C
Allele A
Allele bB
Allele
Allele C
Allele a
Allele b
Allele c
Allele a
Allele Bb
Allele
Allele c
Figure 1.6 Recombination at the gene level, after which the gene sequence at chromosome 1
changes from ABC to AbC. Recombination often involves only part of a gene, which typically leads to
the generation of a unique allele
Comparison between harbour seal and grey seal
Comparison between fin whale and blue whale
Comparison between mouse and rat
Percentage sequence divergence
0.3
0.2
0.1
0.0
1
2
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
Pairwise comparisons of 18 different mitochondrial regions
Figure 1.7 Sequence divergence based on pairwise comparisons of 18 different randomly numbered
regions of mtDNA for i) members of two different genera from the same family: harbour seal (Phoca
vitulina) and grey seal (Halichoerus grypus); ii) members of the same genus: fin whale (Balaenopter
physalus) and blue whale (B. musculus); and iii) members of two different families: mouse (Mus
musculus) and rat (Rattus norvegicus). As we might expect, sequence divergences are highest in the
comparison between families (mouse and rat). However, contrary to what we might expect, the
congeneric whale species are genetically less similar to one another than are the two seal genera. This
is an example of how taxonomic relationships do not always provide a useful guide to overall genetic
similarities. Data from Lopez et al. (1997) and references therein
Source Credit:
Lopez, J. V., Culver, M., Stephens, J. C., Johnson, W. E. and O’Brien., S. J. 1997. Rates of nuclear and
cytoplasmic mitochondrial DNA sequence divergence in mammals. Molecular Biology and Evolution 14:
277–286.
Step 3. DNA extension
Step 2. Annealing primers
Step 3. DNA extension
Step 2. Annealing primers
Step 1. Denaturation
Reverse primer
Step 1. Denaturation
Original DNA sequence
Figure 1.8 The first two cycles in a PCR reaction. Solid black lines represent the original DNA template, short grey lines represent the primers, and hatched lines
represent DNA fragments that have been synthesized in the PCR reaction
Forward primer
Original template DNA
Primer
Newly synthesized DNA
mouse
CGGTCGAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA
frog
CGATCAAACTTGACTATCTAGAGGAAGTAAAAGTCGTAACAAGGTTTCCGTAGGTGAACCTGCGGAAGGA
chicken GAGTGTGAATTGAGTATGTAGAGGAAGTAAAAGTCGTAAGAAGGTTTCCGTAGGTGAACCTGCGGAAGGA
Figure 1.9 Partial sequence of 28S rDNA showing homology in mouse (Mus musculus; Michot et al.,
1982), frog (Xenopus laevis; Furlong and Maden, 1983), and chicken (Gallus domesticus; Azad and
Deacon, 1980). All sites except for those in bold are identical in all three species
Source Credits:
Azad, A. A. and Deacon, N. J. 1980. The 30-terminal primary structure of five eukaryotic 18S rRNAs
determined by the direct chemical method of sequencing. Nucleic Acids Research 8: 4365–4376.
Furlong, J. C. and Maden, B. E. 1983. Patterns of major divergence between the internal transcribed
spacers of ribosomal DNA in Xenopus borealis and Xenopus laevis and of minimal divergence within
ribosomal coding regions. EMBO Journal 2: 443–448.
Michot, B., Bachellerie, J. P., Raynal, F. and Renalier, M. H. 1982. Sequence of the 30 -terminal domain
of mouse 18S rRNA. Conservation of structural features with other pro- and eukaryotic homologs.
FEBS Letters 142: 260–266
Negative electrode
Lane 1
Lane 2
Lane 3
Lane 4
Lane 5
Lane 6
Wells into which
samples are
loaded
Size markers (bases)
500
400
300
200
100
Positive electrode
Figure 1.10 A representation of an agarose gel after DNA fragments have been stained with
ethidium bromide. Lanes 1 and 6 are size markers – note that the smaller fragments migrate through
the gel more rapidly, and therefore further, than the larger fragments, which is why fragments of
different sizes will separate at a predictable rate. The samples in lanes 2 and 5 have a single band of
just over 400 bases long. The sample in lane 3 has two bands that are both close to 200 bases long,
and in lane 4 the two bands are close to 100 and 300 bases long
G
Increase in fragment size (bases)
10
A
T
C
AGGCATCGTA
9
AGGCATCGT
8
AGGCATCG
7
AGGCATC
6
AGGCAT
5
AGGCA
4
AGGC
3
AGG
2
AG
1
A
Figure 1.11 A representation of a sequencing gel. Reactions were loaded into the lanes labeled G, A,
T, and C, depending on which nucleotide was present in the dideoxyribose form. Since the smallest
fragments migrate most rapidly, we can work from the bottom to the top of the gel to generate the
cumulative sequences that are shown on the right hand side