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DNA
DNA (or deoxyribonucleic acid) is the molecule that carries the genetic information in
all cellular forms of life. It belongs to a class of molecules called the nucleic acids,
which are polynucleotides - that is, long chains of nucleotides.
Each nucleotide consists of three components:
a nitrogenous base: cytosine (C), guanine (G), adenine (A) or thymine (T)
a five-carbon sugar molecule (deoxyribose in the case of DNA)
a phosphate molecule
The backbone of the polynucleotide is a chain of sugar and phosphate molecules. Each
of the sugar groups in this sugar-phosphate backbone is linked to one of the four
nitrogenous bases.
What are the components of a molecule of DNA?
DNA is made up of six smaller molecules -- a five carbon sugar called deoxyribose,
a phosphate molecule and four different nitrogenous bases (adenine, thymine,
cytosine and guanine).
RNA
RNA molecules are single stranded nucleic acids composed of nucleotides. RNA plays a
major role in protein synthesis as it is involved in the transcription, decoding, and
translation of the genetic code to produce proteins.
Each nucleotide consists of three components:
a nitrogenous base: cytosine (C), guanine (G), adenine (A) and uracil (U)
a five-carbon sugar molecule (ribose in the case of RNA)
a phosphate molecule
What are the primary functions of RNA?
The three roles of RNA in protein synthesis. Messenger RNA (mRNA) is
translated into protein by the joint action of transfer RNA (tRNA) and the ribosome,
which is composed of numerous proteins and two major ribosomal RNA (rRNA)
molecules.
What is a RNA and what does it do?
Messenger RNA (mRNA) is the RNA that carries information from DNA to the
ribosome, the sites of protein synthesis (translation) in the cell. The coding
sequence of the mRNA determines the amino acid sequence in the protein that is
produced.
Difference
DNA is a double helix whereas RNA is a single helix. DNA is made up of the nucleotides
Adenine (A), Thymine (T), Cytosine (C) and Guanine (G). RNA is also made up of A,
G and C but T is replaced with Uracil (U). Another distinct nucleotide difference between
them is that DNA has one less oxygen on the 5 carbon sugar than RNA; this accounts
for the difference in their names. Deoxyribose simply refers to a ribose sugar lacking an
oxygen molecule.
Gel Electrophoresis
Electrophoresis is the migration of charged molecules, particles or ion in a
liquid medium under the influence of an electric field.
Gel is a cross linked polymer whose composition and porosity is chosen
based on the specific weight and porosity of the target molecules.
Types of Gel:
Agarose gel
Polyacrylamide gel
Objectives
To detect the presence or absence of DNA in the solution after extraction of
DNA from a certain sample
To assess both quality and quantity of DNA samples
Equipments and supplies necessary for conducting agarose gel
electrophoresis :
An electrophoresis chamber and power supply
Gel casting trays, composed of UV transparent plastic
Sample combs, around which molten agarose is poured to form sample wells
in the gel.
Electrophoresis buffer : Tris-acetate-EDTA (TAE) or Tris-borate-
EDTA (TBE).
Loading buffer , which contains something dense (e.g. glycerol) to
allow the sample to “fall” into the sample wells, and one or two
tracking dyes, which migrate in the gel and allow visual monitoring or
how far the electrophoresis has proceeded.
Ethidium bromide , a fluorescent dye used for staining nucleic acids.
NOTE: Ethidium bromide is a known mutagen and should be handled
as a hazardous chemical - wear gloves while handling.
Transilluminator (an ultraviolet light box), which is used to visualize
Ethidium bromide-stained DNA in gels.
NOTE: always wear protective eyewear when observing DNA on a
transilluminator to prevent damage to the eyes from UV light.
4. The sugar phosphate backbone of DNA has a negative charge in neutral to
basic solutions. When we put an electric charge through the sample DNA
will move from a negative pole (cathode) to a positive (anode) pole. The use
of a buffering system stops the solution from becoming acidic.
5. The sample is tagged with ethidium bromide, which becomes fluorescent
under ultraviolet light. Loading dyes are added which make placing the
samples on the gel easier.
6. A photo is usually taken to make a permanent record.
Preparing a standard 1% agarose gel
1. Measure out 1g of agarose.
2. Pour agarose powder into microwavable flask along with 100mL of 1xTAE.
