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Genomes and Molecular Genetics
Molecular genetics
“Primarily concerned with the inter-relationship between the information
macromolecules deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and
how these molecules are used to synthesize polypeptides, the basic
component of all proteins”.
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DNA and RNA molecules are made of a “string”
of covalently linked nucleotides.
Each nucleotide has 3 main components
• A nitrogenous base
• Phosphate group
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Ribose:
Sugars can exist in two optical isomers (enantiomers) called the D- and L-isoforms.
The D-isoforms are the most abundant in biological organisms
D- and L- ribose
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Ribose exists primarily in “cyclic” form
Cyclic formation occurs via an intra-molecular reaction between the anomeric carbon
of the carbonyl group and an alcohol group. Cyclic sugars exist as α (alpha) or β
(beta) “anomers”. Both have different physical and chemical properties
5
β-anomer
2 deoxyribose is the sugar moiety
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in DNA
Nucleotides of DNA and RNA
A SUGAR, nitrogenous BASE and PHOSPHATE group(s) form the nucleotide
building blocks of nucleic acid macromolecules
Purines
Within RNA
molecules thymine
is replaced with
uracil (uridine)
Pyrimidines
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Nitrogen bases of nucleotides can be chemically
modified via enzymes and/or environmental ‘damage’.
Deamination
of adenine
Purines
CH3 Deamination
Pyrimidines of cytosine
Methylation
and
Deamination
Important between
pyrimidines
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Important “modified” cellular nucleotides
Purines
Pyrimidines
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Nucleotide/nucleoside structure and naming conventions:
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“Watson and Crick” complementary base pairing
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Helical forms A, B, and Z-DNA.
B DNA Z DNA
A DNA
right turn Left turn
Right turn
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‘Simple’ in vivo DNA molecules
Plasmids: circular, double-stranded DNA (dsDNA) molecules.
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GENOMES
-The ‘primary’ DNA content of an organism
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Viral nucleic acids
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Bacterial cellular DNA
The “Nucleoid”
Generally a single circular DNA molecule
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Human chromosomes
Humans:
Haploid Ploidy number
(n) = 23
23 chromosomes
-21 autosomes
-2 sex chromosome
Approximately
2 meters of DNA per
nucleus!
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A typical eukaryotic chromosome
Main ‘domains’:
Centromere p-arm
Telomere
Heterchromatin (short)
Euchromatin
q-arm
(long)
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Satellite DNA
highly repetitive “tandem repeat”
DNA sequences
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Satellite DNA
highly repetitive “tandem repeat”
DNA sequences
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Human centromeric DNA largely consists of
various ‘families’ of satellite DNA.
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Minisatellites
90% located at telomeric and sub-telomeric loci
LINE elements
SINE elements
Retrotransposons
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“other” genome-wide repeat sequences
Processed pseudogene:
thought to arise by integration
into the genome of a copy of the
mRNA transcribed from a
functional gene.
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The exons…
(1.5%) of the total DNA content!
50 kb segment of chromosome 12
a) Four genes.
b) 88 genome-wide repeat sequences.
c) Seven microsatellites
d) 30% nongenic, nonrepetitive, single-copy DNA of no known function or significance.
Ultraconserved regions (UCRs)
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Ultraconserved regions (UCRs)
Some regions of the human genome are almost completely
conserved among various species!
UCRs
481 segments longer than
200 base pairs (bp) that are
absolutely conserved
(100% identity with no
insertions or deletions)
between orthologous
regions of the human, rat,
and mouse genomes.
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Studying Genomes
Medically relevant DNA “visualization” methods
in vivo methods:-
Microscopy-
non specific DNA ‘stains’
specific hybridization techniques using labeled ‘probes’
in vitro methods:-
DNA isolation –
specific hybridization techniques using labeled ‘probes’
various types of PCR based DNA sequencing
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visualizing DNA
DNA can be “stained” using chemical dyes that either bind within the double helix
structure or to the double helix-phosphate backbone
DAPI or 4',6-diamidino-2-phenylindole
Hoechst 33342.
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visualizing DNA
General DNA stains can be used to determine A ‘normal’ human male
KARYOTYPES G-banding pattern karyogram:
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visualizing DNA
Nucleic acid hybridization
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visualizing DNA
Nucleic acid hybridization
Melting temperature and hybridization stringency
Probe optimization:
Strand length
(500-800bp)
base composition –
%GC / AT composition
Chemical environment –
the presence of monovalent cations
(e.g. Na+ ions) BSM-4023 2010 36
visualizing DNA
Types of Nucleic acid molecules used in hybridization based
research and diagnosis assays
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Methods of nucleic acid labeling
1) 3)
2)
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Incorporation of labeled nucleotides into “probes”
4) Nick-translation
3) Direct
Chemical Labeling
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In vitro methods of DNA visualization
Southern blot assays
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In vitro methods of DNA visualization
Southern blot assays
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In vitro methods of DNA visualization
Southern blot assays
phosphorimager
Slot blot apparatus DIG labeled and other antibody
detected probes
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In vivo methods of DNA visualization
Fluorescence microscopy
Structure of common fluorophores
Fluorescein-dUTP. Rhodamine
Fluorescence microscopy
The excitation filter barrier “filters” light
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Using fluorescence in situ hybridization to define a translocation breakpoint.
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Summary – background/further reading
Specific chapters for relevant for this lecture:
Basic introduction-
Campbell Biology - Chapter 16: The Molecular Basis of Inheritance
Human Molecular Genetics –Chapter 2
Genomes – Chapter 5
Specific topics -
Human Molecular Genetics Chapter 8 Genome projects and model organisms
Next –
2) DNA replication: The process and regulation of.
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