Forensic Biology by Richard Li

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Forensic Biologyby Richard Li

• Chapter 11 An Introduction to Human Nuclear
  Genome

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Structure Properties of DNA

• 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)

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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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Structure Properties of DNA

• Hydrogen bonds
• AT and GC
• 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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Human Chromsomes

• 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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Human Chromosomes

• 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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Human Nuclear Genome

• 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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Genic DNA

• 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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Genic DNA

• 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

• Intergenic DNA
• Doesnt 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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Human DNA Polymorphisms

• 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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Human DNA Polymorphisms

• 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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Extracting DNA from Cells

• 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)

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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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Organic Extraction

• 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

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Chelex Extraction
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Spin Column Extraction

• 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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Magnetic Beads Extraction

• 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