DNA Techniques€¦ · Recombinant DNA 1) A restriction endonuclease is selected that can cut both...

DNA Techniques

Monday, November 27, 17

Recombinant DNA

Recombinant DNARecombinant DNA: a molecule of DNA composed of genetic material from different sources

Produced in the laboratoryOften combines DNA from different species

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

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.

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

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).

2) The recombinant DNA is introduced into a bacterial host. This is called transformation and requires the correct environmental factors to be met.

3) Bacterial cells enriched with growth medium.

4) Bacterial colonies that have the recombinant DNA is identified using markers.

5) Cells with recombinant DNA selected and grown to produce a larger population.

6) The recombinant DNA is isolated from bacterial cells.

7) An analysis of the recombinant DNA determines of the DNA has been made properly.

Polymerase Chain Reaction

DNA amplification: produces large quantities of DNA for analysis

Polymerase Chain Reaction: automated method of amplifying DNA

1) DNA is heated in a high temperature so DNA is denatured into single strands.

Steps in PCR

2) Once cooled the DNA sample is introduced to nucleotide primers.

Steps in PCR

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

4) Steps 1 to 3 are repeated for several cycles, doubling the amount of DNA each time.

Steps in PCR

Analyzing DNA

Gel Electrophoresis

Gel Electrophoresis: separated fragments of DNA bases on mass and size

Steps in Gel Electrophoresis

1) DNA is added to gel and are died so they can be seen.

2) The gel is placed in a buffer and is electrified. DNA moves to the positive anode because DNA has a negative charge.

3) Smaller DNA fragments move quicker, so travel further. Larger DNA fragments move slower so don’t travel as far.

4) The gel is exposed to UV light exposing bands on DNA

DNA Fingerprinting

DNA Fingerprinting: technology used to identify individuals by analyzing DNA sequences

DNA fingerprinting uses a combination of DNA amplification and electrophoresis.

DNA Techniques€¦ · Recombinant DNA 1) A restriction endonuclease is selected that can cut both...

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Extentofthe DNA SequenceRequired Integration ... · Restriction endonuclease frag-ments were cloned into the M13bacteriophage derivatives mpl8 and mpl9 (30) ... To define the size

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