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DNA Techniques
Monday, November 27, 17
Recombinant DNA
Monday, November 27, 17
Recombinant DNARecombinant DNA: a molecule of DNA composed of genetic material from different sources
Produced in the laboratoryOften combines DNA from different species
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Recombinant DNA
Recombinant DNA is created using restriction enzymes isolated from bacteria
Restriction endonuclease: an enzymes that cuts the interior of DNA in a sequence specific manner
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Steps to Produce Recombinant DNA
1) A restriction endonuclease is selected that can cut both DNA segments.2) Each piece of DNA is reacted with endonuclease.3)Two cute fragments are incubated with DNA ligase which joins the two fragments.
Monday, November 27, 17
Monday, November 27, 17
Gene Cloning
Monday, November 27, 17
Gene Cloning
Gene cloning: The process of manipulating DNA to produce many identical copies of a gene
Scientists clone genes in bacteria that interest them to make them easier to study
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Steps in Gene Cloning
1) A recombinant DNA molecule is produced composed of the gene of interest and a vector(carrier for the gene to be cloned).
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Steps in Gene Cloning
2) The recombinant DNA is introduced into a bacterial host. This is called transformation and requires the correct environmental factors to be met.
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Steps in Gene Cloning
3) Bacterial cells enriched with growth medium.
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Steps in Gene Cloning
4) Bacterial colonies that have the recombinant DNA is identified using markers.
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Steps in Gene Cloning
5) Cells with recombinant DNA selected and grown to produce a larger population.
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Steps in Gene Cloning
6) The recombinant DNA is isolated from bacterial cells.
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Steps in Gene Cloning
7) An analysis of the recombinant DNA determines of the DNA has been made properly.
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Monday, November 27, 17
Polymerase Chain Reaction
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DNA amplification: produces large quantities of DNA for analysis
Polymerase Chain Reaction: automated method of amplifying DNA
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1) DNA is heated in a high temperature so DNA is denatured into single strands.
Steps in PCR
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2) Once cooled the DNA sample is introduced to nucleotide primers.
Steps in PCR
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3) DNA is heated in the presence of Taq polymerase (similar to DNA polymerase but can withstand high temperatures).
Taq polymerase attached free base pairs using complementary pairing.
Steps in PCR
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4) Steps 1 to 3 are repeated for several cycles, doubling the amount of DNA each time.
Steps in PCR
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Monday, November 27, 17
Analyzing DNA
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Gel Electrophoresis
Gel Electrophoresis: separated fragments of DNA bases on mass and size
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Steps in Gel Electrophoresis
1) DNA is added to gel and are died so they can be seen.
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Steps in Gel Electrophoresis
2) The gel is placed in a buffer and is electrified. DNA moves to the positive anode because DNA has a negative charge.
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Steps in Gel Electrophoresis
3) Smaller DNA fragments move quicker, so travel further. Larger DNA fragments move slower so don’t travel as far.
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Steps in Gel Electrophoresis
4) The gel is exposed to UV light exposing bands on DNA
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Monday, November 27, 17
DNA Fingerprinting
DNA Fingerprinting: technology used to identify individuals by analyzing DNA sequences
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DNA fingerprinting uses a combination of DNA amplification and electrophoresis.
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Monday, November 27, 17