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