DNA Technology a Dr. Production

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DNA Technologya Dr. Production
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DNA Coiling
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Replication DNA?DNA

• Occurs during S phase of mitosis in reproducing
  cells only
• DNA template is copied complimentarily and semi
  conservatively
• Multiple oris (in eukaryotes) and replication
  forks form in both directions, producing a
  leading and lagging strand as DNA is elongated to
  the 3 end

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Complimentary Antiparallel

• DNA end with the phosphate attached to the 5
  carbon 5 end
• DNA end with the OH group attached to the 3
  carbon is the 3end
• Synthesis of a n.a. chain always proceeds 5 to
  3

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Okazaki Fragments

• Fragments of DNA 1000-2000bp long
• Produced on the lagging strand
• Must be joined with ligase
• Replication
• DNA Replication Fork Nucleotide Addition
  Videos

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Enzymes

• DNA polymerase?1exonuclease activity, 2 DNA
  repair, 3primary replication enzyme)
• Ligase?joins nucleic acid pieces
• Primase? joins RNA primer to DNA strand
• Helicase?unwinds the DNA
• Nuclease? cuts DNA
• Endonuclease? cuts DNA internally vs at the ends
• Telomerase?catalyzes lengthening of telomeres
• Topoisomerase?alters the degree of supercoiling
  nucleic acids but cutting and rejoining strands

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Mistakes/Mutations

• Mutation? any permanent alteration in the
  sequence of DNA bases
• Point mutations can result in frameshifts in the
  reading frame
• Chromosomal mutations can result in translocations

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Chromosomal Mutations Base-pairing (substitution/
deletion-addition) Mutations
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Mistakes/Mutations

• Silent mutation? change in nt sequence, but no
  change in product
• Nonsense mutation? insertion of stop codon into
  sequence
• Missense mutation? swapping of aa (sickle cell
  anemia, PKU)

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Types of DNA

• Coding DNA
• Less than 5 of the human genome actually codes
  for genes

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• Simple Sequence DNA
• 20-30 human DNA
• Short sequence of 5-10 bp repeat.
• Half of the DNA in a species of crab has
  ATATATATA
• Fruit fly has ACAAACT 12 million times

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• Intermediate-Repeat
• 150-300bp scattered throughout genome, some have
  known functions and code for histones and rRNA
• Huntingtons disease CAG repeats 6-35/38-180bp
  (also various dystrophies)

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Junk DNA ?

• More than 95 percent of all DNA, was called
  "Junk DNA" by molecular biologists, because they
  were unable to ascribe any function to it.
  However that category is becoming smaller as
  scientists identify non-coding DNA as introns,
  transposable elements, pseudogenes, VNTRs
  (variable number tandem repeats and junk.
• Chromosome 11 Flyover

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• Extraneous DNA
• provirus?when the DNA of a virus incorporates its
  self into an eukaryotic cell. Can move around in
  the genome
• reverse transcriptase? enzyme that allows RNA
  strand to be a template of DNA
• transposons? jumping genes

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Where did it come from?

• One hypothesis about the junk is that these
  chromosomal regions are trash heaps of defunct
  genes, sometimes known as pseudogenes, which have
  been cast aside and fragmented during evolution.
• Evidence for a related hypothesis suggests that
  the junk represents the accumulated DNA of failed
  viruses.
• Yet another hypothesis is that the junk DNA
  provides a reservoir of sequences from which
  potentially advantageous new genes can emerge.
  (Junk vs trash)
• As with most things in science, time and
  technology will tell

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Types of RNA

• mRNA messenger RNA Transcribe DNA to RNA
• rRNA ribosomal RNA used in the building of
  ribosomes
• tRNA transfer RNA carry a.a. to ribosome during
  translation
• snRNA small nuclear RNA Mediate the processing
  of DNA transcripts for mRNA, rRNA, and tRNA
• snoRNA small nucleolar RNARNAs within the
  nucleolus have several functions
• miRNAmicro RNA tiny (22 nts) RNA molecules
  that appear to regulate the expression of mRNA
• XIST RNA. inactivate one of the two X chromosomes
  in female vertebrates.
• gRNA guide RNA found in mitochondrial DNA
• sl RNA splice leader RNA 5mRNA cap
• hnRNA heterogenous nuclear RNA nonspliced or
  immature RNA

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Isolating DNA

• Restriction enzymes/ endonucleases? cut at
  specific palindromic sequences
• 5 G/AATT C 3
• 3 C TTAA/G 5
• cleavage leaves sticky ends
• G AATTC
• CTTAA G
• Steps in Cloning a Gene
• DNA Restriction Step by Step

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Recombination

• Recombination?process in which one or more
  nucleic acid molecules are rearranged or combined
  to produce a new nucleotide sequence
• In eukaryotes, usually occurs as the result of
  crossing-over during meiosis
• In prokaryotes, usually occurs as the result of
  plasmid DNA
• Construction of a Plasmid Vector
• Mechanism of Recombination
• DNA Transformation

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RFLP Analysis

• Restriction Fragments. Those are the fragments of
  DNA that were cut by restriction enzymes.
• Length, and refers to the length of the
  restriction fragments.
• Polymorphism, a Greek term that literally means
  "many shapes". The lengths of some of the
  restriction fragments differ greatly between
  individuals, thus there are many shapes, or
  lengths, of DNA possible.
• Molecular biologists have identified regions of
  the human genome where restriction fragment
  lengths are highly variable between individuals.
  These regions are called RFLP markers.

VNTR Tutorial
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Electrophoresis

• Move DNA through a matrix with a sieving effect
• Agarose gels for DNA
• PAGE for proteins
• Single digest uses 1 enzyme
• Double digest produces 2 restriction patterns
• Interactive Animation, Simplified

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Blotting

• Goes from the gel to filter paper to make and
  autoradiograph
• Southern blot uses RNA to probe DNA strands
  (SB2)
• Northern blot uses DNA to probe RNA strands
• Western blot immunoblotting uses pp-antibody
  binding reaction

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Hybridization

• These hybridization reactions can be used to
  detect and characterize nucleotide sequences
  using a particular nucleotide sequence as a
  probe.
• used to find relatedness in n.a.

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Polymerase Chain Reaction (PCR)
PCR2 PCR3 PCR4 PCR Rap PCR Song
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Transcription DNA?mRNA

• Occurs continually throughout the cell cycle
• Occurs in the nucleus as messenger RNA copies DNA
• cDNA

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• Operonpromoter operator TFs
• Promoter region, upstream of gene, signals start
  of transcription
• Transcription factors mediate binding of RNA
  polymerase
• Operon is controlled by regulatory genes such as
  repressor genes

Control in Prokaryotes http//trc.ucdavis.edu/bio
sci10v/bis10v/week6/08lacoperon.html
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Transcription Initiation Complex
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Splicing of introns exons

• Non-coding regions (introns) of mRNA are spliced
  out of the mRNA strand before leaving the nucleus
• 5cap is methylated and 3tail is polyadenylated
  to indicate orientation at the ribosome

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Translation mRNA?protein

• A?amino acyl (initiation with Met.)
• P?peptide bond (elongation)
• E?exit (termination with UAG, UAA, UGA)

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Regulatory Proteins

• Lac Operon
• Lac Operon Induction
• Regulatory Proteins Repression

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Signal Transduction

• Signal Transduction Pathway
• My Dog is Broken (A Case Study for Cell
  Signaling)

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GMOs

• Classical vs Transgenic Breeding
• Harvest of Fear (What about this fish?)
• Babies by Design?
• Transgenic Mouse

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Gene processing
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Resources

• BioStudio Animations http//www.biostudio.com/a_s
  itemap.htm
• Biology Animations http//science.nhmccd.edu/biol
  /ap1int.htm
• Restriction Mapping Tutorial
• DNA Replication
• BioZone BioLinks http//www.biozone.co.nz/CELL_BI
  OL_AND_BIOCHEM.html
• Lac Operon Animation
• Lac Operon Induction
• Biointeractive Animations
• Animated Tutorials Molecular Biology
• Processing of Gene Information Animation
  Prokaryotes vs Eukaryotes
• Learners TV
• Fundamentals of Molecular Diagnostic Links
• Gene Expression
• DNA Technology Quiz