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Chapter 11: An Introduction to Human
Nuclear Genome
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DNA = Deoxyribonuleic acid
 Linear polynucleotide consisting of four types of
nucleotide monomers
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Each nucleotide contains:
 Deoxyribose sugar, a Nitrogenous base, and a
Phosphate group
 Four nitrogenous bases:
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Adenine (A)
Cytosine (C)
Guanine (G)
Thymine (T)
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H group only,
no OH group
Deoxyribose sugar
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Purines (2 rings)
Pyrimidines 1 ring)
Nitrogenous bases of DNA. (a) adenine, (b) guanine, (c) cytosine, (d) thymine
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Phosphodiester
bond
Deoxyribopolynucleotide chain
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Two deoxypolynucleotides hydrogen bond to one another
in an anti-parallel fashion to form the DNA double helix
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Hydrogen bonds
 A::T and G:::C
 Individually weak, collectively strong
 Can be “melted” by enzymes or heat to denature
the double helix into two single deoxypolynucleotide strands
 If DNA heated, cool slowly and strand renature
(come back together)
 Reversible melting curve
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Most human cells carry 46 DNA molecules
 23 from mother, 23 from father
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DNA molecules are wrapped around proteins
and tightly packaged to form chromosomes
 Short arm (p) & Long arm (q)
 Centromeres- DNA sequences found near the
point of attachment of mitotic or meiotic spindle
fibers
 Telomeres- ends of chromosomes
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Gametes- spermatozoa and ova
 Haploid (one complete copy of genome)
 22 autosomes + 1 sex chromosome = 23
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Somatic Cells- most other cells except
reproductive
 Diploid (one copy of genome from each parent)
 Two copies of each autosome + 2 sex
chromosomes= 46
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Karyogram of human genome
22 autosomes and 2 sex
chromosomes = 24
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Human karyotype = 2
sets of 23 each = 46
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Human genome
 3.2 billion base-pairs
 25,000 genes (40% of genome)
▪ Encode information for the synthesis of proteins
▪ Function of about 50% have been identified
 Lots of non-coding (intergenic) regions (60%)
▪ Structural function, junk, and evolutionary debris
 Human Genome Project
▪ Initiated in 1990
▪ Now mostly complete
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Genes are transcribed into RNAs
 mRNAs: Translated into polypeptides (which fold
and may also combine with other polypeptides to
form functional proteins)
▪ Proteins carry out almost all activities/functions of the
cell
▪ Structures
▪ Enzymes
▪ Signaling molecules
 rRNAs, tRNAs, other small functional RNA
molecules
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Structure of a typical gene
 Cis-regulating sequences
▪ Ensure polypeptide or functional RNA is produced in the
right cell type at the right time and for the right length
of time; Enhancers and Silencers
 Promoter
▪ Recruits RNA polymerase to gene so that sequence can
be transcribed to RNA
 Untranslated regions
 Exons and introns
 3’ transcription termination sequence
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Intergenic DNA
 Doesn’t code for polypeptides or functional RNAs
 Some has structural role; most no known function
 Includes single copy and repetitive DNA
 Repetitive DNA
▪ Interspersed repeats
▪ SINEs, LINEs, LTR
▪ Tandemly repeated DNA
▪ Satellite DNA
▪ Minisatellites
▪ Microsatellites
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Interspersed repetitive DNA
Tandem repetitive DNA
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Differences between individual genomes
 Sequence polymorphisms
▪ E.g. AACTCTGG versus AACCCTGG
 Length polymorphisms
▪ E.g. AACTCTGG versus AACTCTCTGG
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DNA markers
 Polymorphisms among people
▪ Genetic mapping
▪ Forensic DNA profiling
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Alternative forms of DNA polymorphisms are
called alleles
 Since humans are diploid can be
▪ Heterozygous (two different alleles)
▪ Homozygous (two of the same allele)
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Genotype = alleles carried by an individual
Phenotype = physical and behavioral
characteristics of an individual
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~1 in 100 bp DNA different
32 million differences total
99% identical
Chimps and
humans share
about half their
DNA with
bananas
50% identical
~1 in 1,000 bp DNA different
3.2 million differences total
99.9% identical
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General steps:
 Lyse open cells
 Separate DNA from all other cell components
(e.g. small molecules, lipids, polysaccharides,
proteins
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Lysis usually achieved by treating cells with
SDS (detergent) and proteinase K
 PK also degrades proteins into amino acids
 DTT may also be needed (sperm, hair)
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Several common methods:
 Organic extraction
▪ Advantage: Yields high quality DNA
▪ Disadvantages: Toxic and time-consuming
 Chelex extraction
▪ Advantage: Very fast
▪ Disadvantage: Poor separation of DNA from other cell
components
 Spin column extraction
▪ Advtantage: Yields high quality DNA
▪ Disadvantage: Toxic
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Lyse cells with SDS/PK /(DTT)
Add equal volume of phenol /chloroform/
isoamyl alcohol
Vortex and centrifuge
Remove aqueous layer
Add more phenol
Repeat procedure
Concentrate by ethanol precipitation or over
size exclusion column
Aqueous layer
Phenol layer
Add tissue, swab, or swatch
Discard tissue, swab, or swatch
Close lid, vortex,
centrifuge
95 degrees C
20 minutes
Centrifuge
Remove
Chelex®
(10%)
Supernatant into
new tube and retain
Figure 7-1. Chelex method for extracting DNA from cells or biological swabs or stains.
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Lyse DNA with SDS/PK/(DTT)
Add chaotropic salts
• Dehydrate DNA
Place onto column
• Column has silica membrane (+++)
• Column has size exclusion
properties
• Silica binds tightly to dehydrated
DNA
• Centrifuge
• Small molecules flow through
• Molecules not strongly
negatively charged flow through
• Elute DNA in low salt buffer
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Lyse DNA with SDS/PK/(DTT)
Add chaotropic salts
• Dehydrate DNA
Place in tubes with magnetic beads
• Beads coated with silica (+++)
• Silica binds tightly to dehydrated
DNA
• Place in magnetic stand
• Beads sucked to side to tube
(along with DNA)
• Remove supernatant
• Elute in low salt buffer
• Place in magnetic stand
• Remove supernatant