Lab Techniques

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Lab Techniques

• James Chappell Cheuk Ka Tong

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Contents Page

• Restriction Enzymes
• Gel Electrophoresis
• Blotting techniques-Southern, Northern and
  Western
• DNA sequencing
• Polymerase Chain Reaction (PCR)
• Recombinant DNA
• Gene Cloning
• References/ Recommended Reading

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Restriction Enzymes

• Restriction Nuclease - An enzyme that cleaves a
  molecule of DNA at any site where a specific
  short sequence of nucleotides occurs.
• 2key types
• Endonuclease- Cleaves within the DNA molecule
• Exonuclease- Cleaves at the ends of the DNA
  molecule

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Endonucleases

• 4 types - classified on subunit composition,
  cleavage position, sequence-specify and co-factor
  requirement.
• Type II is the main one that is used in gene
  cloning.
• Two key terms
• Recognition sites Nucleotide sequence that is
  recognised.
• Cleavage sites- Phosphodiester bond that is
  cleaved.

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Endonucleases

• Make break the phosphodiester bond of each of the
  stands of the double helix.

5----G-3 5AATTC----3 3----CTTAA--5
3G----5
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Endonucleases-Recognition site Cleavage site
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Endonucleases-Recognition site

• 3 Considerations-
• Sequence Determine specificity
• Length of sequence- Determines frequency
• Palindrome- sequence that reads the same
  backwards and forwards.
• Isoshizomers Restriction enzymes that recognise
  the same recognition site

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Endonucleases-Cleavage site
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EndonucleasesApplications

• Allow specific cutting and removal of genes from
  a complex molecule of DNA.
• Complementary sticky ends (cohesive ends) allow
  joining of DNA molecules.
• "The work on restriction nucleases not only
  permits us easily to construct recombinant DNA
  Molecules and to analyze individual genes but
  also has led us into the new era of synthetic
  biology where not only existing genes are
  described and analyzed but also new gene
  arrangements can be constructed and evaluated"
• Nobel prizes and restriction enzymes in GENE
  (1971)

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Gel Electrophoresis

• Electrophoresis - the migration of charged
  molecules in an electric field though a solution
  or solid support
• Various types defined by support used
• Paper amino acids, small peptides
• Polyacrylamide Proteins, small DNA/RNA (lt500bp)
• Agarose DNA/RNA
• Good preparative and analytical method

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Gel Electrophoresis

• Gel electrophoresis uses a cross-linked polymers
  (agarose) that contain various pores.
• Pores allow molecular sieving, where molecules
  e.g. DNA, can be separated based upon there
  mobility through the gel.

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DNA Gel Electrophoresis

• Mobility Charge Molecular Dimensions

• Charge per nucleic acid is constant
• This means separation is based upon length of the
  DNA molecules and this is how we can separate and
  identify DNA molecules.

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DNA Gel Electrophoresis

• Linear DNA has a linear relationship to distance
  migration.
• If add molecular markers of known mass can
  calculate mass of our fragment by plotting a
  linear plot.

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DNA Gel Electrophoresis

• Other factors determining mobility-
• Polymer concentration e.g. Agarose
• Conformation of DNA
• Electrophoresis

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DNA Gel Electrophoresis

• Detection
• Dye e.g. ethidium bromide
• Audioradiography 32P,
• Blotting (see later)
• Uses
• Analytical- Can determine size of DNA fragment,
• Preparative Can identify a specific fragment
  based on size

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Blotting Techniques

• Blotting Transfer of DNA, RNA or Proteins,
  typically from a electrophoresis gel to a
  membrane e.g. nitrocellulose. This membrane can
  then be subject to further techniques such as
  hybridization.
• Hybridization Process where two complementary
  single strands of nucleic acid (DNA or RNA) form
  a double helix.

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Blotting Techniques

• Using specific probes that are labelled specific
  sequences of DNA can be identified.
• There are three main hybridization techniques
  which vary in the sample blotted and the probes
  used
• Northern Blot-Transfer of an RNA sample separated
  and identified using DNA or RNA probes.
• Southern Blot-Transfer of an DNA sample separated
  and identified using DNA or RNA probes.
• Western Blot- Transfer of an Protein sample
  separated and identified typically using an
  antibody.

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Applications

• The main use of this technique is to identity any
  changes in DNA sequencing or genes expressed,
  e.g. comparing genes expressed by a diseased cell
  to genes expressed by an healthy cell.
• Other uses include- Testing for hereditary
  disease, Evolutionary history of species,
  Screening e.g.food supply
• Applications to synthetic biology
• - identification of various parts in natural
  organisms,
• -?more?

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DNA Sequencing

• DNA Sequencing Determining the order of
  nucleotides in a DNA molecule
• Key technique as it can give us information about
  a DNA molecule, e.g. location and order of genes,
  restriction sites.
• In addition, for recombinant DNA gives
  verification of gene cloning experiments.
• 2 possible uses for project Identify sequence
  of new part, - Checking recombinant
  DNA.

• Various techniques are available.
• The key technique used today is Dideoxy method.

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DNA Sequencing
Deoxyribonucleotide acid This is essentially the
monomer of DNA. Polymerization of nucleotides
occurs by condensation reaction of a 5 phosphate
to a 3 hydroxyl group
Dideoxyribonucleotide acid There is no 3hydroxyl
group to allow polymerization.
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Polymerase Chain Reaction (PCR)

• A method for amplifying specific DNA sequences.
• Components required
• - Target sequence
• - A pair of primers
• - dNTPs (ATGC)
• - DNA polymerase

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PCR

• One PCR cycle involves three steps
• - Strand separation (95ºC)
• - Hybridization of primers (54ºC)
• - DNA synthesis (72ºC)
• After n cycles, the sequence is amplified 2n-fold.

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Polymerase Chain Reaction (PCR)

• Five noteworthy features of PCR
• The sequence of the target need not be known.
• The target can be much larger than the primers
  (gt10 kb).
• Primers do not have to perfectly match flanking
  sequences.
• Stringency can be controlled by temperature and
  salt (MgCl2).
• PCR is very sensitive.

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Recombinant DNA Technology

• The construction of new combinations of unrelated
  genes.
• These novel combinations can be cloned and
  amplified by introducing them into host cells.

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Recombinant DNA Technology

• A DNA fragment of interest is covalently joined
  to a DNA vector.
• - A vector can replicate autonomously in an
  appropriate host.
• - Plasmids and phage ? are common vectors for
  cloning in E.coli.

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Recombinant DNA Technology

• The DNA fragment of interest and the plasmid
  vector are both cut using the same restriction
  enzyme.
• The single-stranded ends of the fragment are
  complementary to those of the cut plasmid.
• The DNA fragment and the cut plasmid are annealed
  and then joined by DNA ligase.

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Gene Cloning

• One of the most useful plasmids for cloning is
  pBR322.
• pBR322 contains genes for resistance to
  tetracycline and ampicillin.
• Different endonucleases can cleave this plasmid
  at a variety of unique sites.

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Gene Cloning

• Insertion of DNA at the EcoRI site does not alter
  either of the genes for antibiotic resistance.
• However insertion at the SalI or PstI site causes
  insertional inactivation.
• ? Basis for selection of cells containing
  recombinant DNA.

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References/Recommended Readings

• Chapter 8 Manipulating Proteins, DNA and RNA
• - Molecular Biology of the Cell (4th Edition),
  Alberts, Johnson, Lewis, Raff, Roberts, Walter,
  Garland Science
• 2. Chapter 6- Exploring Genes
• -Biochemistry (5th Edition), Berg, Tymoczko,
  Stryer, Freeman