What is the principle of DNA isolation

DNA isolation and plasmid isolation


DNA can easily be isolated from eukaryotic and prokaryotic cells. The isolation of DNA is the first step in the working process of a gene cloning. The cloning of a gene in turn is the prerequisite for its further analysis, because by cloning a DNA can be obtained and reproduced in pure form.

An overview of the individual gene cloning steps:



  • Isolation of DNA (genomic or cDNA) and plasmid DNA
  • Cut the DNA and plasmid with a restriction enzyme
  • Ligation of DNA fragments with the plasmid DNA
  • Transformation of bacteria with recombinant plasmid
  • Selection of bacterial clones which have taken up a plasmid
  • Isolation of the clone with the searched DNA fragment


Video: Principle of gene cloning (2D animation); Excerpt from the video "Cloning".
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DNA cloning: why?


Cloning of Genomic DNA
The total DNA from cells of the same species is cut into pieces and each piece is cloned. The result is a collection of DNA fragments that represent the entire genome of these cells: the genomic DNA library. It is suitable for isolating genes (with promoters, introns etc.) and intergenic DNA sequences.

Cloning of cDNA
The total mRNA from cells of the same species is isolated, transcribed into cDNA and each cDNA is cloned. The result is a collection of cDNA fragments which represent the entire mRNA of these cells: the cDNA library. It is suitable for isolating DNA sections that code for proteins. Since the coding sequences of a genome usually only represent a few percent of the total DNA of a cell, cDNA banks are much smaller (fewer clones) than genomic banks and a desired DNA sequence (coding for protein) can therefore be found with less effort .


DNA isolation


Genomic DNA
The most important steps:
  • Break open cells. Physical: e.g. crushing the frozen cells in a mortar. Chemical: e.g. cell lysis with detergents
  • Centrifugation of insoluble cell debris
  • Purifying the DNA. Breakdown of proteins with proteases. Denaturing and precipitation of the proteins with
  • Phenol. The nucleic acids remain in solution. Separation of the insoluble and soluble fraction by centrifugation. Ribonuclease treatment to break down RNA.
  • Enrichment of the DNA by means of ethanol or isopropanol precipitation

Details: chemistry of nucleic acids


Video: Principle of DNA isolation (2D animation); Excerpt from the video "DNA isolation".
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Video: isolating cell nuclei; Excerpt from the video "DNA isolation".
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Video: nuclear lysis; Excerpt from the video "DNA isolation".
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Video: cleaning with phenol; Excerpt from the video "DNA isolation".
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Video: DNA Aggregation; Excerpt from the video "DNA isolation".
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cDNA
cDNA (complementary DNA) is an "imprint" of the mRNA and corresponds to the DNA which codes for a sought-after mRNA.

The most important steps:
  • Isolation of all RNAs of a cell in which the desired gene is expressed, i.e. the protein is synthesized.
  • Purification of mRNA (i.e. separating tRNA and rRNA) by affinity chromatography
  • Synthesis of a complementary DNA strand on the mRNA template: -> RNA-DNA double strand
  • Breakdown of the RNA strand
  • Synthesis of a complementary DNA strand on the DNA template: -> cDNA (DNA double strand)

Action:
  • Isolation of total cellular RNA (80% rRNA, 15% tRNA, 5% mRNA)
  • Separating a fraction which mainly contains mRNA. Virtually every mRNA molecule has a sequence of adenine nucleotides = poly (A) tail at its 3 'end. This opens up a very convenient way of separating mRNA from the rest of the cell RNA. Deoxythymidine oligonucleotides ("Oligo (dT)") are bound to cellulose and filled into a column. If the entire cell RNA is allowed to run through this column, the mRNAs with their poly (A) tails "stick" to the oligonucleotides (formation of A-T base pairs), while the remaining RNAs pass through the column. A low ionic strength buffer is now allowed to run through the column. As a result, the poly (A) -oligo (dT) hybrids dissolve and the purified mRNA flows out of the column.
  • cDNA synthesis. Short deoxythymidine oligonucleotides are added to the purified mRNA. These hybridize with the poly (A) tails and then act as primers for reverse transcriptase, an enzyme from retroviruses. With the help of this enzyme, mRNAs are first overwritten via RNA-DNA double strands into single-stranded DNAs and then into double-stranded DNAs. Like genes, cDNAs contain the information about the formation of proteins, but unlike genes, they do not have introns.




Plasmid isolation


Plasmids are minichromosomes that multiply autonomously. They are called vectors
Vectors are vehicles for transporting DNA into a host cell. By inserting a foreign gene into the vector, a recombinant DNA molecule is constructed which can replicate in the host cell.


Plasmids

In addition to chromosomal DNA, bacteria (prokaryotic cells) often also harbor small (5 to 1000 kilobase pairs, kb) circular DNA molecules, plasmids, which are replicated and transcribed by the host cell independently of the genomic DNA. Resistance plasmids are medically important: they contain genes that make the host cell insensitive to antibiotics.

In recombinant DNA technology, artificially modified plasmids with the following elements are used:




Origin of replication: It is for the replication of the plasmid. This is where the bacterial replication complex begins with bidirectional replication.

Selectable marker genes: They enable the selection of bacterial cells that have taken up the plasmid (e.g. resistance genes).

Restriction enzyme interfaces: they are important for the insertion of foreign DNA. Such an interface should only occur once in a plasmid.

Plasmids can be made in the laboratory as needed. Artificially modified plasmids often contain so-called "polylinkers". These are interfaces for various restriction enzymes connected in series.


Bacteriophages as vectors

Somewhat larger DNA segments can be cloned when using the bacteriophage lambda as a vector. The bacteriophage Lambda has a very effective mechanism to introduce its 48,502 base pair DNA into a bacterium.

The general procedure for cloning DNA in bacteriophage lambda relies on two key features of the bacteriophage lambda genome:

  • About a third of the bacteriophage lambda genome can be dispensed with and replaced by foreign DNA.
  • DNA is only packed into infectious phage particles if it is 40,000 to 50,000 base pairs in length.

Bacteriophage lambda vectors have been developed which can easily be split into three pieces; two of them contain essential genes and together have a length of about 30,000 base pairs. Therefore, to generate "viable" phage particles, additional DNA must be inserted between them. Bacteriophage lambda vectors can clone DNA fragments up to 23,000 base pairs in length, and their design ensures that all viable phage particles contain a foreign DNA fragment. Once the fragments of bacteriophage lambda have been linked to foreign DNA fragments of appropriate size, the resulting recombined DNA can be packaged into phage particles by adding them to raw bacterial cell extracts containing all of the proteins needed to assemble a complete phage. This process is called in vitro packaging. The bacteriophage vector is now prepared for the insertion of the recombined DNA into E. coli cells.



Cosmids as vectors

Cosmids are recombined plasmids that incorporate useful features of both the plasmids and the bacteriophage. They have been developed for the cloning of even larger DNA fragments.
used in cloning.

Action:
  • Grow plasmid-containing bacteria in a liquid medium.
  • Treat bacteria with lysozyme to destabilize the cell wall.
  • Lyse bacteria with detergent and denature bacterial DNA and proteins in alkaline pH.
  • Neutralize the homogenate. Proteins and bacterial genomic DNA precipitate and remain in solution.
  • Separate the plasmids from the precipitated material by centrifugation.
  • Extract the plasmids with organic solvents and precipitate with alcohol.


Video: Principle of plasmid isolation (2D animation); Excerpt from the video "Plasmid Isolation".
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Video: isolate bacteria; Excerpt from the video "Plasmid Isolation".
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Video: Separate plasmid DNA; Excerpt from the video "Plasmid Isolation".
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Video: Purify Plasmid DNA; Excerpt from the video "Plasmid Isolation".
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Why is the bacterial genomic DNA denatured and the plasmids remain in solution?
solution

Solution Both the bacterial genomic DNA and the plasmid DNA are circular, but differ in size. During cell lysis, the bacterial genomic DNA is broken down or linearized. During denaturation, the linear DNA is separated into single strands. The much smaller plasmid DNA remains circular and can therefore not be denatured.

Video: Separate plasmid DNA; Excerpt from the video "Plasmid Isolation".
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