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DNA Technology

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DNA Extraction

• Chemical treatmentsChemical treatments cause cells and nuclei to burst

•The DNA is inherently stickysticky, and can be pulled out of the mixture

•This is called “spooling”“spooling” DNA

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“Spooled” DNA

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Cutting DNACutting DNA• Restriction enzymesRestriction enzymes cut

DNA at specific sequences• Useful to divide DNA into

manageable fragmentsmanageable fragments

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• These cuts usually occur These cuts usually occur in the following forms:in the following forms:

1.The cut can be made 1.The cut can be made straight across a base-straight across a base-pair sequence resulting in pair sequence resulting in a a "Blunt End“"Blunt End“

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2. The cut can be made in 2. The cut can be made in an offset manner leaving an offset manner leaving exposed nucleotide exposed nucleotide sequences. These sequences. These exposed sequences are exposed sequences are called called "Sticky Ends""Sticky Ends"

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Gene SplicingGene Splicing• The presence of sticky ends The presence of sticky ends

allows segments of DNA to be allows segments of DNA to be joined together. joined together.

• Recombinant DNA Recombinant DNA – joining of – joining of DNA from 2 or more speciesDNA from 2 or more species

• DNA strands which have been DNA strands which have been cut by the same restriction cut by the same restriction enzyme can easily bond enzyme can easily bond together according to base together according to base pairing rules. pairing rules.

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Gene splicing cont’

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Gene splicing use:• Allows genes to be "cut & pasted"

between organisms. – E.g. production of human insulin.

• The DNA sequence of insulin is identified, & cut out with restriction enzyme.

• A plasmid from E. coli is removed & cut open using the same restriction enzyme

• Both fragments have complimentary sticky ends, they bind, the gene for human insulin is integrated into plasmid

• The plasmid is reinserted into bacterial cell which produces insulin.

• Human insulin can now be extracted and provided to diabetics.

PlasmidPlasmid – – a ring of a ring of DNA found DNA found in a in a bacterium bacterium in addition in addition to its main to its main strand of strand of DNADNA

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Electrophoresis

•DNA can be separated based on size & chargesize & charge

•The phosphate phosphate groupsgroups are negativelynegatively charged

•DNA is placed in a gelgel and electricityelectricity is run through

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Electrophoresis machine

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Electrophoresis• Negative DNANegative DNA moves toward

the positive end• SmallerSmaller fragments move

farther and faster through farther and faster through porespores

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Electrophoresis

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Tutorial with animation

• http://www.tvdsb.on.ca/westmin/science/sbioac/genetics/Electro.htm

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Animations

• http://www.lewport.wnyric.org/jwanamaker/animations/Chrom%26Elpho.html

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Steps in DNA Steps in DNA SequencingSequencing

• Many Many copiescopies of a single of a single strand of DNA are placed strand of DNA are placed in a test tubein a test tube

• DNA polymeraseDNA polymerase is added is added• A mixture of nucleotides is A mixture of nucleotides is

added some of which have added some of which have dye molecules attacheddye molecules attached

• Each base (A,T,C,G) has a Each base (A,T,C,G) has a different color dyedifferent color dye

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Steps in DNA Steps in DNA SequencingSequencing

•By chance, some some dyed nucleotidesdyed nucleotides & some regular onessome regular ones are added

•Dye molecules are large and stop the stop the chainchain from growing

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DNA DNA SequencingSequencing

•The result is DNA fragments of multiple sizes multiple sizes with colors with colors that can be that can be identifiedidentified

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DNA SequencingDNA Sequencing

• After the gel After the gel separates the separates the resulting fragments resulting fragments by size, we by size, we 'read' the 'read' the sequence from sequence from bottom to topbottom to top..

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DNA FingerprintingDNA Fingerprinting• DNA is now a powerful tool in identification.DNA is now a powerful tool in identification.• Based on the fact that the amount of "junk DNA" Based on the fact that the amount of "junk DNA"

differs uniquely between individuals.differs uniquely between individuals.• Structural genes are often separated by large regions Structural genes are often separated by large regions

of repeating base pairs.of repeating base pairs.• The number of these repeats is unique to an The number of these repeats is unique to an

individual.individual.• When DNA from a person is cut with a restriction When DNA from a person is cut with a restriction

enzyme, the length of the fragments will be unique to enzyme, the length of the fragments will be unique to that individual.that individual.

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Fingerprinting cont’• This will produce a unique banding pattern This will produce a unique banding pattern

following a gel electrophoresis.following a gel electrophoresis.• This test is highly accurate, and the This test is highly accurate, and the

probability of another individual possessing probability of another individual possessing an identical banding pattern is estimated as an identical banding pattern is estimated as around 1:14,000,000,000.around 1:14,000,000,000.

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Fingerprinting cont’

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Copying DNACopying DNA• Polymerase Chain ReactionPolymerase Chain Reaction• Also called PCR • A method of making many copies

of a piece of DNA

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Steps in Copying Steps in Copying DNADNA

•A DNA molecule is placed in a small test tube

•DNA polymeraseDNA polymerase that can work at high temps is added

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Steps in Copying Steps in Copying DNADNA

•The DNA is heatedDNA is heated to separate the two strands

• PrimersPrimers, short pieces of DNA complementary to the ends of the molecule to be copied, are added

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Copying DNACopying DNA

•The tube is cooled, and DNA DNA polymerase polymerase adds new adds new bases to the bases to the separated separated strandsstrands

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PCRPCR

Large amounts of DNA can be Large amounts of DNA can be made from a small starting made from a small starting

samplesample

PCR Virtual Lab

• http://learn.genetics.utah.edu/content/labs/pcr/

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CloningCloning• CloneClone- a member

of a group of genetically identical cells

• Produced by asexual asexual reproductionreproduction (mitosis)

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Cloning Cloning organismsorganisms

• A body cellbody cell from one organism and an egg cellegg cell from another are fused

• The resulting cell divides divides like a normal embryolike a normal embryo

(many identical cells produced)

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Cloning “Dolly”Cloning “Dolly”

GEL ELECTROPHORESIS VIRTUAL LAB

• http://learn.genetics.utah.edu/content/labs/gel/

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REFERENCES -

http://www.accessexcellence.org/RC/VL/GG/plasmid.php