Chapter 11: An Introduction to Human Nuclear Genome.
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Transcript of Chapter 11: An Introduction to Human Nuclear Genome.
Chapter 11: An Introduction to Human Nuclear Genome
DNA = Deoxyribonuleic acid Linear polynucleotide consisting of four
types of nucleotide monomersEach nucleotide contains:
Deoxyribose sugar, a Nitrogenous base, and a Phosphate group
Four nitrogenous bases:▪ Adenine (A)▪ Cytosine (C)▪ Guanine (G)▪ Thymine (T)
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Deoxyribose sugar
H group only, no OH group
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Nitrogenous bases of DNA. (a) adenine, (b) guanine, (c) cytosine, (d) thymine
Purines (2 rings)
Pyrimidines 1 ring)
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Deoxyribopolynucleotide chain
Phosphodiester bond
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Two deoxypolynucleotides hydrogen bond to one anotherin an anti-parallel fashion to form the DNA double helix
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 deoxy-polynucleotide 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
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
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 genome22 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
Differences between individual genomes Sequence polymorphisms
▪ E.g. AACTCTGG versus AACCCTGG Length polymorphisms
▪ E.g. AACTCTGG versus AACTCTCTGG 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)
Genotype = alleles carried by an individual
Phenotype = physical and behavioral characteristics of an individual
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~1 in 100 bp DNA different32 million differences total
99% identical
~1 in 1,000 bp DNA different3.2 million differences total
99.9% identical
Chimps and humans share about half their
DNA with bananas
50% identical
General steps: Lyse open cells Separate DNA from all other cell
components (e.g. small molecules, lipids, polysaccharides, proteins
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)
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
Phenol layer
Aqueous layer
Lyse cells with SDS/PK /(DTT)Add equal volume of phenol
/chloroform/ isoamyl alcoholVortex and centrifugeRemove aqueous layerAdd more phenolRepeat procedureConcentrate by ethanol precipitation
or over size exclusion column
Figure 7-1. Chelex method for extracting DNA from cells or biological swabs or stains.
Discard tissue, swab, or swatch
95 degrees C
20 minutes
Chelex®
(10%)
Add tissue, swab, or swatchClose lid, vortex,
centrifuge
Centrifuge
Remove
Supernatant into new tube and
retain
• 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
• 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