gel electrophoresis: the method for separation and purification of nucleic acids and proteins
TRANSCRIPT
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GEL ELECTROPHORESIS:
The method for separation and purification of nucleic acids and
proteins.
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Electrophoresis of Onion DNA
• The purpose of this lab is to first isolate onion DNA by lysis and then separate and isolate the individual nucleic acids of the onion DNA by gel electrophoresis.
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THEORY
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PART ONE:
What is DNA?
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BASES
• DNA is composed of what are called bases known as purines and pyrimidines.
• Each of these purines and pyrimidines are heterocyclic amines, therefore are basic in nature.
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BASES OF DNA
Purines
ADENINE
GUANINE
Pyrimidines
CYTOSINE
THYMINE
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ADENINE
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GUANINE
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CYTOSINE
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THYMINE
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HOW DO THESE BASES ‘HOOK’ TOGETHER?
• It takes the additional bonding of a sugar and phosphate group.
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THE SUGAR OF DNA
2-DEOXY-D-RIBOSE
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2-DEOXY-D-RIBOSE
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SUGAR + BASE
• The monosaccharide sugar 2-deoxy-D-ribose bonds with the purine base through carbon number 1 on the sugar and nitrogen number 9 on the base.
• This linkage of a sugar and a base is known as a nucleoside.
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ADDITION OF THE PHOSPHATE GROUP
When the phosphoric acid forms a bond with the CH2OH it forms a phosphate ester
with the nucleoside.
The result is what is known as a nucleotide.
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BASE + SUGAR + PHOSPHORIC ACID
NUCLEOTIDE
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The Big Picture
• These nucleotides bond together to form a chain.
• This chain is called a nucleic acid which is the backbone of DNA.
• The bases of one nucleic acid chain bonds with the bases of another nucleic acid chain.
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Although……
• In the bonding of bases in a nucleic acid, a pyrimidine must be opposite a purine.
• For example:– Thymine + Adenine
– Cytosine + Guanine
The formation of these base pairs is done by hydrogen bonding, which in turn hooks two nucleic acid chains together.
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Part 2:
What is Gel Electrophoresis?
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GEL ELECTROPHORESIS
• Gel electrophoresis is used to separate proteins and in the case of this lab, nucleic acids, by relying on the movement of charged particles in an electric field.
• The medium used is agorose gel submerged in a buffer solution.
• A positive electrode is placed on one side of the buffer solution and a negative on the other.
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Gel Electrophoresis Cont…..
• The buffer solution should be either more acidic or more basic than the isoelectric point of the nucleic acid in question.
• This determines toward which electrode the nucleic acid will migrate in the arogose gel.
• If the nucleic acid’s PH corresponds to that of the buffer, there will be no net movement.
• more acidic negative electrode
• more basic positive electrode
• Equal no net movement
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MOVEMENT
• Because a nucleic acid incorporates different side chains two different nucleic acid chains will have slightly different net charges at a particular PH.
• Thus, their movement in the agorose gel will be different and electrophoresis can be used to separate different nucleic acids.
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Nucleotides, at a PH of 7.4, ionizes the phosphate links between each nucleotide.
This gives the DNA fragments a negative charge and causes them
to migrate to the positively charged electrode.
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Size is also a determining factor of how far a nucleic acid will migrate in the agorose gel.
Larger polynucleotides move more slowly through the gel than
smaller ones.
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SODIUM DODECYL SULFATE (SDS)
• SDS is used in gel electrophoresis because it binds to the nucleic acid causing it to unfold into a ‘rod like’ shape. Therefore, all of the nucleic acids because they have similar shapes, will tend to travel at rates proportional to their chain lengths.
• Also, the SDS molecule ensures that the nucleic acids are negatively charge and that they will migrate toward the positive electrode.