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Nucleic Acid • DNA
• Contains the biological instructions that make each species unique
Biotechnology Techniques • Made of _____________________, which are linked covalently
into a polynucleotide chain with __________________________
from which the bases extend.
Reymund M. Mabbagu, RMT, MSMT • DNA does not usually exist as a single molecule, but instead as
_______________________ that are held tightly together in the
shape of a ___________________.
• Each DNA sequence that contains instructions to make a protein is
known as ____________________.
Reymund M. Mabbagu, RMT, MSMT
Nucleic acid Extraction and Purification
• The DNA extraction process frees DNA from the cell and then
separates it from cellular fluid and proteins so you are left with pure
DNA.
• Basic Steps:
1. _________________________– to expose the DNA within
2. _________________________ – salting out procedure
3. _________________________ – using PCR
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
Purification and Detection of Nucleic Acid
• Cell lysis • Gel electrophoresis
1. Remove membrane lipids by adding a detergent • Based on the motion of
2. Remove proteins by adding a protease
charged particles in electric
3. Precipitate the DNA with an alcohol
field.
• Salting out Procedure
• Used to simplify the deproteinization procedure by S.A. Miller et.al.
• Involves salting out of the cellular proteins by dehydration and precipitation.
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
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• Types of Electrophoresis: • Materials used in Electrophoresis
1. Slab Gel Electrophoresis • Buffer
2. Disc Electrophoresis • Gel
3. Isoelectric Focusing Electrophoresis • Loading dye
4. Isotachophoresis
• Visualization dye
5. Pulse-field Electrophoresis
6. Capillary Electrophoresis • Ladder
7. Microchip Electrophoresis
8. Two-dimensional (2D) electrophoresis (isoelectric focusing and sodium
dodecyl sulphate)
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
• Buffer • Gels
• To carry the current and • Agarose gel
______________ during electrophoresis • Polysaccharide polymer extracted from
•A solution of seaweed
_________________________________
_________________________________ • Polyacrylamide gel
• pH is constant as it takes up or release • Acrylamide in combination with cross-
protons linker methylene bisacrylamide
• Tris buffers: Tris borate EDTA, Tris • Best for very small DNA fragments and
single stranded DNA
phosphate EDTA, Tris acetate EDTA
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
• Loading dye • Visualization Dye
• Tracking dye with density agent • Intercalating dyes:
• Ethidium bromide
• Tracking dye: bromthymol blue, xylene • Under excitation with UV light at 300 nm,
cyanol EtBr in DNA emits visible light at 590 nm
• _______________________________________ • Carcinogenic!
• Usually runs ahead of the smalles fragment of
DNA
• SYBR Green
• Produces orange band in UV light
• Density agent: Ficoll, sucrose, glycerol
• _______________________________________________
• 25 – 1,000x more sensitive than EtBr
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
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• Gel electrophoresis Procedure:
1. A sample is applied to a supporting
medium
2. An electric current is passed through the
• Ladder medium to achieve the desired
separation.
• A set of standard that are used to 3. Polymeric gels are used as supporting
identify the approximate size of a media
molecule run on a gel during 4. The gel are prepared and cast as a
electrophoresis continuous cross-linked matrix (the
crosslinking give rise to the pores, and
• Principle: Mol.wt. is inversely the choice of agaraose versus
proportional to migration rate polyacrylamide gels depends on the size
through a gel matrix. of the molecules to be separated
• Agarose
• Polyacrylamide
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
• The charge on the molecules to be • Other Separation technique for DNA and RNA detection
separated leads them to move • Radioactive labelling
through the gel toward an electrode
of the opposite charge.
• Autoradiography
• The separation are based on the size
and the sieving action of the gel.
• Chemiluminiscent
• The Smaller oligonucleotide the
farther it moves than the larger one.
• Fluorescence
Shown are DNA fragments from six samples run on a gel,
stained with a fluorescent dye and viewed under UV light.
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
PCR
• Developed in 1983 by Kary B. Mullis
Polymerase Chain Reaction • a chemical reaction that molecular biologists use
to amplify pieces of DNA. This reaction allows a
single or a few copies of DNA to be replicated into
Reymund M. Mabbagu, RMT, MSMT millions or billions of copies.
• To make a huge number of copies of a gene
necessary to have enough starting template for
sequencing
Reymund M. Mabbagu, RMT, MSMT Reymund M. Mabbagu, RMT, MSMT
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• Basic Components needed for PCR: • Applications of PCR:
1. DNA source –Blood, tissues, organs, stool and others • DNA or RNA labelling
• DNA or RNA cloning
2. Primers – Starting point for DNA synthesis • DNA and RNA detection
• Drug discovery
3. Nucleotide bases
• DNA and RNA quantitation
4. Enzyme – DNA polymerase; Taq polymerase • Genotyping and DNA-based identification
• Thermus aquaticus (1976)
5. Co-factor for DNA polymerase
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