DNA Extraction and Purification

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Extraction of [[DNA|DNA]] from a source organism(animal,plants or microbes) is easier when compared to using the same technique to isolate RNA.This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in Genomic DNA isolation are;
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Extraction of [[DNA|DNA]] from a source organism (animal, plants or microbes) is easier when compared to using the same technique to isolate [[RNA|RNA]]. This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in [[genomic DNA|genomic DNA]] isolation are:
  
1) Disruption of cell membranes(animal cells)/cell walls(plant cells)  
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#Disruption of cell membranes ((animal cells/cell walls (plant cells))
  
 
This can be done using two approaches;  
 
This can be done using two approaches;  
  
A) Physical Approaches  
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==== A) Physical Approaches ====
  
i) Sonication: the process of directing vibration generated from a mechanical process towards a substance(e.g DNA). [[Bacteria|Bacteria]] cells containing DNA are subjected to sonication to break the cell membranes and release the DNA proteins. An equipment known as a Sonicator is used for this process.  
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#'''Sonication:''' the process of directing vibration generated from a mechanical process towards a substance(e.g DNA). [[Bacteria|Bacteria]] cells containing DNA are subjected to sonication to break the cell membranes and release the DNA proteins. An equipment known as a Sonicator is used for this process.
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#'''Shearing:''' this is the accidental tearing apart of long DNA molecules during laboratory preparations. It may also be done intentionally.
  
ii) Shearing: this is the accidental tearing apart of long DNA molecules during laboratory preparations. It mayu also be done intentionally.
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==== B) Chemical Approaches   ====
  
B) Chemical Approaches 
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This is the use of [[Enzymes|enzymes]] such as [[Lysozyme|lysozyme]] and detergents on cell components or cytosolic domains to cause lysis of the cells. [[Lysozyme|Lysozyme]] is often added to detergents in this process because of its ability to digest cell wall components of [[Gram-positive|gram-positive]] bacteria
  
This is the use of [[Enzymes|enzymes]] such as [[Lysozyme|lysozyme]] and detergents on cell components or cytosolic domains to cause lysis of the cells. Lysozyme is often added to detergents in this process because of its ability to digest cell wall components of gram-positive bacteria
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#Low speed centrifugation of isolated cells to remove debris
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#Addition of [[RIBONUCLEASE|Ribonuclease]] to destroy [[RNA|RNA]]. This enzyme Ribonuclease does not destroy DNA. 
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#Removal of contaminants by mixing with equal volumes of [[Phenol/chloroform|phenol/chloroform]]. This denatures proteins and they are precipitated to form a blue interface layer in the test tube. Phenol and chloroform are not miscible in water. 
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#Concentration of DNA by precipitation
  
2) Low speed centrifugation of isolated cells to remove debris
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This is done by adding 2 volumes of [[Ethanol|ethanol]] plus 0.2 M [[Sodium Chloride|NaCl]].
  
3) Addition of [[RIBONUCLEASE|Ribonuclease]] to destroy RNA
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Measurement of DNA concentration is carried to ensure if protein is pure/ free of protein.
  
4) Removal of contaminants by mixing with equal volumes of phenol/chloroform. This denatures proteins and they are precipitated to form an interface layer in the test tube.
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This can be done by using either of these two methods;
  
5) Concentration of DNA by precipitation
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#Absorbance method 
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#Flourescence method
  
This is done by  adding 2 volumes of ethanol plus 0.2M NaCl.  
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However,the simplest is the absorbance method which is carried using [[Spectrophotometry|Spectrophotometry]]. This uses a simple laboratory equipment called Spectrophotometer including UV lamp, transparent curvettes and a solution of purified DNA. DNA absorbs UV light maximally at 260nm and proteins absorb light maximally at 280nm. This means that DNA absorbs light at 260nm 1.8 times more strongly than at 280nm. If there is by protein contaminants in the purified DNA, the absorbance at 280nm increases. In this case, equal volumes of phenol/ chloroform is added and the following procedure is repeated.<br>
  
6) Measurement of DNA concentration<br>
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This pure DNA has high stability and can be stored frozen for a long duration of time.
 
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This can be done by using either of these two methods;
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i) Absorbance method&nbsp;
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ii) Flourescence method
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&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;However,the simplest is the absorbance method which is carried using [[Spectrophotometry|Spectrophotometry]]. This uses a simple laboratory equipment called Spectrophotometer including UV lamp,transparent curvettes and a solution of purified DNA. Absorbance readings is taken at 260nm,this is because DNA absorbs light more at this wavelength.
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Latest revision as of 03:42, 24 October 2014

Extraction of DNA from a source organism (animal, plants or microbes) is easier when compared to using the same technique to isolate RNA. This is because DNA is very stable and is the same in all cells of multicellular organism. The key steps in genomic DNA isolation are:

  1. Disruption of cell membranes ((animal cells/cell walls (plant cells))

This can be done using two approaches;

A) Physical Approaches

  1. Sonication: the process of directing vibration generated from a mechanical process towards a substance(e.g DNA). Bacteria cells containing DNA are subjected to sonication to break the cell membranes and release the DNA proteins. An equipment known as a Sonicator is used for this process.
  2. Shearing: this is the accidental tearing apart of long DNA molecules during laboratory preparations. It may also be done intentionally.

B) Chemical Approaches 

This is the use of enzymes such as lysozyme and detergents on cell components or cytosolic domains to cause lysis of the cells. Lysozyme is often added to detergents in this process because of its ability to digest cell wall components of gram-positive bacteria

  1. Low speed centrifugation of isolated cells to remove debris
  2. Addition of Ribonuclease to destroy RNA. This enzyme Ribonuclease does not destroy DNA. 
  3. Removal of contaminants by mixing with equal volumes of phenol/chloroform. This denatures proteins and they are precipitated to form a blue interface layer in the test tube. Phenol and chloroform are not miscible in water. 
  4. Concentration of DNA by precipitation

This is done by adding 2 volumes of ethanol plus 0.2 M NaCl.

Measurement of DNA concentration is carried to ensure if protein is pure/ free of protein.

This can be done by using either of these two methods;

  1. Absorbance method 
  2. Flourescence method

However,the simplest is the absorbance method which is carried using Spectrophotometry. This uses a simple laboratory equipment called Spectrophotometer including UV lamp, transparent curvettes and a solution of purified DNA. DNA absorbs UV light maximally at 260nm and proteins absorb light maximally at 280nm. This means that DNA absorbs light at 260nm 1.8 times more strongly than at 280nm. If there is by protein contaminants in the purified DNA, the absorbance at 280nm increases. In this case, equal volumes of phenol/ chloroform is added and the following procedure is repeated.

This pure DNA has high stability and can be stored frozen for a long duration of time.

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