DNA the GENE

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DNA the GENE
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History of DNAs Discovery

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• 1. Griffith Experiment demonstrates the
  Transformation of bacteria DNA is later found
  to be the transforming principle

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2. Hershey-Chase demonstrate DNA is hereditary
material not proteins by using radioactive
isotopes
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3. Meselson-Stahl demonstrate the
Semiconservative Replication of DNA using
radioactive nitrogen
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Discovery of DNA

• Frederick Griffith
• Was studying Streptococcus Pneumonia
• Smooth vs. Rough Strains
• Smooth had a mucous coat and were pathogenic
  (caused pneumonia)
• Rough were non-pathogenic
• Conducted an experiment with mice
• Found out that the Rough bacteria became
  transgenic with the Smooth and killed the mouse

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Discovery of DNA

• Avery, McCarty and MacLeod
• What was the genetic material in Griffiths
  experiment?
• Purified the heatkilled S-bacteria
• Into DNA, RNA, and Protein
• Mixed each with the R cells to see which one
  transformed

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Discovery of DNA

• Hershey-Chase Experiment
• Studied viruses that infect bacterial cells
  called Bacteriophages
• Viruses use Bacteria to multiply
• Protein or DNA responsible for multiplying within
  the bacteria
• Tagged the Protein with radioactive S
• Why?
• Tagged the DNA with radioactive P
• Why?
• Checked the Virus Progeny for Radioactive Elements

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P
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P
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Discovery of Structure
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The Structure of DNAa double helix?

• Rosalind Franklin and Maurice Wilkins use X-Ray
  diffraction to view structure

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Watson and Crick propose a double helix.
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From Chromosomes to Genes
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DNA Basic Composition

• DNA is made up of nucleotides
• Nucleotides are made of
• ...Deoxyribose sugar
• Phosphate
• Base
• bases are guanine,cytosine, thymine and adenine

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The Structure of DNAa double helix?

• Chargaffs Nucleic Acid Ratios
• Measured the base compositions of several species
• Percentage of each base present
• Human DNA
• A 30 and T 29
• G 20 and C 19

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DNA The Deoxyribose Sugar
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DNA The Phosphate
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DNA The Nitrogenous Bases

• Purines
• Adenine and Guanine
• Double Ring Structure
• Pyrimidines
• Thymine and Cytosine
• Single Ring Structure

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Single Stranded DNA Nucleotides can only be
added to the 3 end of the nucleotide and
therefore addition of new nucleotides is always
5-----gt 3 DNA is anti-parallel!!
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Why do they pair up?

• Double helix had a uniform diameter
• Purine Purine
• too wide
• Pyrimidine Pyrimidine
• too narrow
• Purine Pyrimidine
• fits the x-ray data

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Purines

• Adenine
• Guanine
• All double ring structures

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DNA BASE PAIRS
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Pyrimidines

• Cytosine
• Thymine
• single ring

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How does it know to pair up?

• ADENINE ALWAYS PAIRS WITH THYMINE
• Two hydrogen bonds
• GUANINE ALWAYS PAIRS WITH CYTOSINE
• Three hydrogen bonds

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DNA STRUCTURE
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One last look
Why does it twist?
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DNA Replication
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Why must DNA Replicate?

• Species Survival
• DNA must replicate BEFORE cell division
• Synthesis during Interphase
• All genes must be present in the daughter cells

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DNA ReplicationNumber the following events 1-5

• Enzymes unwind and unzip
• Hydrogen bonds break, forming bubbles
• Free nucleotides in the nucleus start
• process of complementary base pairing
• Nucleotides are fused together by DNAPolymerase
  only 5 to 3
• Results in two identical double helixes

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How does DNA Replicate?

• Enzymes unwind and unzip
• Hydrogen bonds break, forming bubbles
• Free nucleotides in the nucleus start process of
  complementary base pairing
• Nucleotides are fused together by DNA Polymerase
  only 5 to 3
• Results in two identical double helixes

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How does DNA Replicate?
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How does DNA Replicate?
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DNA Structure
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Single stranded
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Nucleotides can only be added to the 3 end of
the nucleotide and therefore addition of new
nucleotides is always 5-----gt 3
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replication
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Leading strand is continious
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Lagging strand is done in segments as each primer
is added only after a coding segment is exposed.
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Replication Steps

• DNA polymerase and helicase enzymes start the
  following
• DNA uncoils and unzips exposing the DNA template
  and free nucleotides
• bases pair A-T and G-C as new strand is added in
  a 5 to 3 direction
• Two identical strands of DNA are made

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replication3
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DNA and RNA functions

• Replication, Transcription, and Translation

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function of DNA
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RNA Nucleotides

• Made of the following
• Ribose sugar
• Phosphate
• one of four bases ( uracil replaces thymine)

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Types of RNA

• M RNA- messenger RNA carries the DNA
  instructions(gene) out of nucleus to ribosome
• tRNA-transfer RNA carries amino acids to their
  appropriate location during protein synthesis (
  gene expression )
• r RNA - ribosomal RNA makes up much of the
  ribosome and is essential to translation

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Transcription Steps

• DNA uncoils and exposes template
• RNA nucleotides base pair with DNA template A-U,
  G-C via RNA polymerase
• This new mRNA is then processed and leaves the
  nucleus to be translated.

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Transcription
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Promoter Regions direct Transcription
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Processing Genetic Material

• After transcription mRNA is PROCESSED
• INTRONS ARE DELETED
• A CAP AND TAIL IS ADDED(G-p-p-p)5
• poly a tail AAA-AAA
• start and stop codon

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DNA Processing
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Translation Steps

• Messenger RNA is at the ribosome and the tRNA
  nucleotides will base pair A-U, G-C
• The tRNA has the amino acid attached to it and
  when it finds the right codon the RNA anticodon
  places the amino acids in their proper sequence
  for protein synthesis
• The bond that forms between two amino caids is
  called a peptide bond.
• Base ( Uracil replaces Thymine)

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45 different anticodons exist AUG is initiation
codon GTP supplies energy intiator tRNA carries
methionine small ribosomal unit
attaches intiiation factors-proteins bring all
parts together
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• Aminoacyl-tRNA syntase matches each amino acid to
  the correct tRNA

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Steps in Translation

• Initiation
• Elongation-elongation factors enable addition of
  tRNAs to A site.
  codon recognition
  peptide bonds form

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• translocation -tRNA move from A site to P site
• Termination-UAA, UAG UGA stop the process

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Polyribosomes- clusters of ribosomes translating
the same mRNA
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At the ribosome
Growing polypeptide
P
A
tRNA molecule exits
Next amino acid to be added
tRNA molecules with amino acids
P site--- peptidyl-tRNA binding site
A site- aminoacyl-tRNA binding site
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Gene Expression

• Various cells express different genes
• Organization of chromatin controls expression
• Regulation of expressed genes occurs at each step
• Control of transcription is most important
  regulatory mechanism ( binding factors and
  enhancers)
• some binding factors are sensitive to hormones

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Transposons

• Stretches of DNA that can move from one location
  to another ( Jumping genes)

DNA
transposase
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Immunoglobulin genes undergo permanent rearrangeme
nt during antibody production.
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DNA TECHNOLOGIES

• SEQUENCING-determine order of bases
• PCR (polymerase chain reaction)-makes repeated
  copies of desired DNA
• RFLPS(restriction fragment length polymorphs)
  -unique gene fragments used as a fingerprint
• Gel Electrophoresis- separate DNA by size on a
  gel bed
• Probes- Radioactive tags label DNA

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PCR-polymerase chain reaction

• Makes several copies of DNA
• adjust temperature and enzyme
• addition of nucleotides with DNA polymerase

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Prepare single strand complimentary DNA
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Mix hybrid (Known fragment as Hybrid )
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Hybrid 3
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Blot on Nylon Film
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Use probe to identify position of gene on a
chromosome
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PROBES
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Reverse Transcriptase

• Viral enzyme
• transcribes DNA from RNA
• if you know the protein you can dtermine the mRNA
  which makes the protein
• revers the transcription process to make DNA

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Sequencing Methods

• Chain termination- Sanger Method
• Restriction enzymes specific
• Restriction fragments vary in size
• gel electrophoresis resolves the fragments

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Chain Termination Method-uses dedeoxynucleotides
that terminate the synthesis of DNA strands at
specific bases
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Electrophoresis
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Restriction Analysis
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RFLP restriction fragment length polymorph
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RFLPS

• Restriction fragments are created by cutting DNA
  with enzymes that cut at specific locations and
  create fragments of various size. These
  fragments can then be amplified and separated by
  gel electrophoresis

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DNA Fingerprints
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VNTRvariable number tandem repeats
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Restriction Analysis
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Other Technologies

• Recombinant DNA - gene splicing
• Transgenic organism- an organism that contains
  another organisms DNA

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Recombinant DNA

• Plasmid DNA
• Ligase enzyme Bacterial Cell
• Restriction Enzyme Bacterial cell wall
• Host cell Sticky ends
• Vector
• DNA fragment desired gene to be cloned

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Transgenic Organism
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Genetic Therapy