How do we analyze DNA?

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How do we analyze DNA?

• Gel electrophoresis
• Restriction digestion
• Sequencing

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DNA analysis

• How do we manipulate DNA to improve our crop?
• First we need to identify which genes in the DNA
  sequence are important for the trait we are
  trying to improve
• BUT - DNA from a single human cell can be over 2m
  in length! Some plants have genomes as large as
  7.5 x 1010 base pairs (25 times the size of the
  human genome).

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Gel Electrophoresis

• 1949 a team led by chemist Linus Pauling found
  two samples of hemoglobin from healthy and
  sickle-cell anemia sufferers migrated at
  different rates.
• Today, gel electrophoresis is indispensable
• Wide variety of applications includes
  determination of a gene's sequence, isolation of
  entire chromosomes, and separation and
  characterization of proteins

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Uses outside the laboratory

• DNA fingerprinting evidence
• Men proving or disproving paternity via the
  technique
• Hospitals replacing conventional heel prints with
  genetic fingerprints as a means of identifying
  newborns.

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How does it work?

• Agarose and polyacrylamide are the two media most
  commonly used in gel electrophoresis. Both
  substances create a porous matrix through which
  charged macromolecules migrate in response to an
  electric field.
• Negatively charged DNA, for example, travels
  toward the positively charged electrode when
  current is applied.

http//www.life.uiuc.edu/molbio/geldigest/photo.ht
ml
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• Pores in the matrix limit the migration of large
  molecules, while smaller molecules migrate more
  freely and travel farther toward the opposite
  pole. This molecular sieving separates molecules
  on the basis of size.
• Agarose is used as the support matrix to separate
  nucleic acids and very large proteins or
  complexes.
• Agarose is a natural polysaccharide derived from
  certain types of red seaweed. When heated and
  then cooled, agarose solidifies into a solid
  matrix with relatively large, nonrestrictive
  pores.

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• Agarose gel electrophoresis (AGE) can be used to
  separate molecules by charge or by their
  molecular weight.
• One of the most common applications of AGE is its
  use in separation of the fragments generated by
  cleaving DNA with restriction enzymes
• Gel electrophoresis not only separates
  macromolecules, but also allows the researcher to
  actually use the nucleic acid or protein by
  transferring it to a support membrane made of
  nitrocellulose or nylon, and then probing it with
  radioisotope- or enzyme-labeled complementary DNA
  or antibodies

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• DNA has to be cut into more manageable pieces
  before it can be analyzed
• Restriction enzymes cut DNA in very specific
  places that are determined by the order of 4-8
  base pairs of nucleotides
• These smaller pieces can then be cloned into a
  plasmid or bacteriophage vector for amplification
  and further analysis

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Restriction Mapping

• A restriction map is a description of
  restriction endonuclease cleavage sites within a
  single piece of DNA
• First step in characterizing an unknown DNA, and
  a prerequisite to manipulating it for other
  purposes
• Restriction enzymes that cleave DNA infrequently
  (e.g. those with 6 bp recognition sites) and are
  relatively inexpensive are used to produce a map

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Creating a Map - Digestion with Multiple
Restriction Enzymes

• Digest samples of the plasmid with a set of
  individual enzymes, and with pairs of those
  enzymes
• Digests are then "run out" on an agarose gel to
  determine sizes
• Deduce where each enzyme cuts, which is what
  mapping is all about

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e.g. Plasmid with 3000 base pair (bp) fragment of
unknown DNA

• Digestion with Kpn I yields two fragments 1000
  bp and "big
• Digestion with BamH I yields 3 fragments 600,
  2200 and "big
• double digest yields fragments of 600, 1000 and
  1200 bp (plus the "big" fragment)

http//arbl.cvmbs.colostate.edu/hbooks/genetics/bi
otech/enzymes/maps.htmltop
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• If the process outlined above were conducted with
  a larger set of enzymes, a much more complete map
  would result.
• In essence, single digests are used to determine
  which fragments are in the unknown DNA, and
  double digests to order and orient the fragments
  correctly.

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DNA Sequencing

• Short stretches of DNA can be sequenced using
  primers that are based on the known nucleotides
  where the restriction enzyme has cut the DNA
• DNA heated to a critical temperature called the
  Tm will denature or separate into two single
  strands
• These strands can be used to build new
  complementary strands, or can reanneal or stick
  back together when the temperature is reduced

http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html
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• Any single-stranded piece of DNA can only
  hybridize with another if their sequences are
  complementary. If we have just one strand, we can
  actually build another strand to match it.

• Polymerase Chain Reaction (PCR)
• For each strand, we provide a primer, which is a
  short piece of DNA that sticks to one end of the
  strand.

http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html

• DNA polymerase "reads" the bases on one strand
  and can attach the complementary base to the
  growing strand.

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• DNA sequencing reactions are just like the PCR
  reactions for replicating DNA
• The reaction mix includes the template DNA, free
  nucleotides, an enzyme (usually a variant of Taq
  polymerase) and a 'primer'

http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html
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• A small proportion of each of the four bases in
  the reaction mixture is specially modified to
  form a dideoxynucleotide and is labeled with a
  unique fluorescent dye or tag
• As the strand is replicated it will stop
  elongating each time one of these
  dideoxynucleotides is added

http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html
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DNA Sequencing

• As the reaction proceeds to build a new DNA
  strand from the existing one the signal from each
  of the bases can be recognized and so we can work
  out in which order the bases were added

http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html
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http//seqcore.brcf.med.umich.edu/doc/educ/dnapr/s
equencing.html

• A 'Scan' of one gel lane The computer reads
  the lane for us! This is what the sequencer's
  computer shows us - a plot of the colors detected
  in one 'lane' of a gel (one sample), scanned from
  smallest fragments to largest. The computer even
  interprets the colors by printing the nucleotide
  sequence across the top of the plot

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How do we use this information?

• We can compare organisms to one another
• DNA fingerprints allow for identification of each
  individual
• Once genes have been identified we can begin to
  work with these areas of the genome in order to
  improve traits