Study Guide to Dr Hill

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http//ca.youtube.com/watch?v7PvnyAEC65Aeurlhtt
p//genomics.xprize.org/
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Dr. Kathleen Hill khill22_at_uwo.ca Office Hours
Monday noon to 5pm Room 333 Western Science
Centre Hill Research and Teaching
Website http//www.uwo.ca/biology/Faculty/hill/ind
ex.htm Study Guide for 2581b WebCT site
281b Dr. Hill
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What is the function of soap, salt and alcohol in
DNA extraction?
Iron Grad Recipe
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In the laboratory a common method of DNA
extraction uses SDS (soap) to soluabilize
membranes and inactivate proteins and Proteinase
K to digest proteins and then extractions with
Phenol Chloroform to physically remove proteins
from DNA. Salt and alcohol permit precipitation
of DNA
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Recombinant DNA TechnologyKey Concepts
Two key properties of nucleic acids
ACGT TGCA
Complementary
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5
Antiparallel
ACGT TGCA
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3
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Recombinant DNA TechnologyKey Concepts
A key property of Proteinnucleic acid
interactions
Sequence specific binding
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Restriction Endonucleases Bind to Specific
Palindromic DNA Sequences

• Palindrome (Rotational symmetry)
• Cuts through covalent bonds in the sugar
  phosphate backbone of DNA
• Blunt/flush -Blunt ends
• Staggered -Single stranded sticky ends
• 3 overhang
• 5 overhang

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Fig. 9.2
Certain restriction endonucleases cleave the
covalent bonds of the sugar phosphate backbone at
sites in the palindromic sequence that
create Blunt or flush ends
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Fig. 9.2.b
Certain restriction endonucleases cleave the
covalent bonds of the sugar phosphate backbone at
sites in the palindromic sequence that create 5
sticky or cohesive ends
Hydrogen bonds possible Hybridization possible
with complementary sequence
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Fig. 9.2.c
Certain restriction endonucleases cleave the
covalent bonds of the sugar phosphate backbone at
sites in the palindromic sequence that create 3
sticky or cohesive ends
Hydrogen bonds possible Hybridization possible
with complementary sequence
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• Restriction Endonucleases
• derived from prokaryotes
• have the Genus and species name of their
  prokaryote origin
• numbered since there may be many of these enzymes
  in a single species

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• Restriction Endonucleases are derived from
  prokaryotes
• Restriction Endonucleases cut double stranded DNA
  at specific palindromic sequences
• Bacteria use restriction enzymes to protect from
  invading viral nucleic acids
• Bacteria methylate their DNA to protect it from
  digestion by their own restriction enzymes

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Fig. 9.3
Determining the frequency that a palindrome is
present in a DNA sequence
Note Above The assumption was made that A, C,
T, G occur with equal frequencies in the DNA
target sequence
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Fig. 9.3
Note you may be asked to determine the
average restriction fragment size in cases where
there are nonequivalent frequencies of the four
nucleotides
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Fig. 9.3
Restriction Endonucleases with shorter
recognition sequences tend to have more frequent
cut sites and produce shorter fragments
Frequent cutter
Less frequent cutter
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Fig. 9.3.a

1. The average fragment length for a four base
   cutter is 256 bp
2. The average fragment length for a six base cutter
   is about 4kb

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Restriction Endonuclease Digestion
EcoRI

• Cutting DNA
• Circular DNA with one cut one fragment
• Linear DNA with one cut two fragments

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Fig. 9.4.a
The concept of a complete vs. partial digest
Complete Digest All possible sites cut in all
templates in the reaction
Partial Digest The reaction is incomplete and
not all sites and templates are cut
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• A linear DNA molecule has two target sites for
  restriction enzyme AvaI. What is the maximum
  number of DNA lengths that can be produced if the
  sample is only partly digested?
• A. 3
• B. 4
• C. 5
• D. 6
• E. 9

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• A linear DNA molecule has two target sites for
  restriction enzyme AvaI. What is the maximum
  number of DNA lengths that can be produced if the
  sample is only partly digested?
• A. 3
• B. 4
• C. 5
• D. 6
• E. 9

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• A linear DNA molecule has two target sites for
  restriction enzyme AvaI. What is the maximum
  number of DNA lengths that can be produced if the
  sample is only partly digested?
• Question variables to be aware of
• Linear or circular
• Number of target sites
• Type of digest partial or complete

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Restriction Fragment Analysis
Gel electrophoresis separates DNA fragments
primarily on the basis of size/length
Restriction Enzyme digest of plasmid DNA
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Fig. 9.5.a
DNA is electrophoresed through a polymer
Agarose vs. Acrylamide Gels Agarose larger
migration space for DNA Polyacrylamide smaller
migration space for DNA
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Fig. 9.5.a

• DNA is loaded (pipetted) into the wells of the
  gel
• Sucrose or glycerol provide density so the DNA
  sample sinks into the wells of the submerged gel
• A dye helps to see the sample fall into the well

DNA is negatively charged and migrates at a rate
relative to its size/length
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Fig. 9.5.b.2
Anatomy of a DNA Gel
-
Ethidium Bromide is a dye that intercalates with
DNA and fluoresces upon UV exposure
DNA Migration
Size markers assist in determining fragment
length
Ethidium Bromide Migration

UV Light
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Fig. 9.5.b.2
Genomic DNA after Digestion
Cutting a complex DNA sample with a frequent
cutter results in a smear Size markers assist
in determining fragment length
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