Chapter 11: An Introduction to Human Nuclear Genome

 DNA = Deoxyribonuleic acid  Linear polynucleotide consisting of four types of nucleotide monomers  Each nucleotide contains:  Deoxyribose sugar, a Nitrogenous base, and a Phosphate group  Four nitrogenous bases: ▪ Adenine (A) ▪ Cytosine (C) ▪ Guanine (G) ▪ Thymine (T) 2

3 Deoxyribose sugar H group only, no OH group

4 Nitrogenous bases of DNA. (a) adenine, (b) guanine, (c) cytosine, (d) thymine Purines (2 rings) Pyrimidines 1 ring)

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6 Deoxyribopolynucleotide chain Phosphodiester bond

7 Two deoxypolynucleotides hydrogen bond to one another in 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 8

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10  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 13

14 Karyogram of human genome 22 autosomes and 2 sex chromosomes = 24

15 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 17

 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 18

 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 19

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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 22

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24 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 25

 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 26

27 ~1 in 100 bp DNA different 32 million differences total 99% identical ~1 in 1,000 bp DNA different 3.2 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 alcohol  Vortex and centrifuge  Remove aqueous layer  Add more phenol  Repeat procedure  Concentrate 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 swatch Close 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