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Gene Cloning Notes

The basic steps of gene cloning are

  1. Insert a DNA fragment containing a gene to be cloned in a vector (a circular DNA molecule). This produces a recombinant DNA molecule.
  2. The vector transports the gene into a host cell.
  3. The vector multiplies along with the cloned gene in the host cell.
  4. When the host cell divides, copies of recombinant DNA are passed to the daughter cells

The gene introduced through the vector is now said to be cloned.

Source

Gene Cloning & DNA Analysis by T. A. Brown

What is PCR?

Unlike gene cloning, Polymerase Chain Reaction is carried out in a test tube. DNA is mixed with reagents and the test tube is placed in a thermal cycler. Thermal cycler enables the mixture to be incubated at a series of temperatures that are varied in a preprogrammed manner.

Following are the steps:

  1. Heat the mixture to 94 degree Celsius to break H bonds that hold the double strand together. This is called denaturation.
  2. Cool the mixture to 50-60 degrees. The strands can join back together at this temperature but they won't due the heavy presence of oligonucleotides or primers which anneal to the DNA molecules at specific positions.
  3. Temperature is raised to 74 degrees, which is optimum temperature for Taq DNA polymerase which is present in the mixture. Taq DNA polymerase attaches to one end of each primer and synthesizes new strands of DNA, complementary to the template DNA molecule. Thus we now have 4 strands instead of 2.
  4. We then increase the temperature back to 94 degrees and denature to repeat the process. At the end of this step, we would have 8 DNA strands. We continue until, the DNA is sufficiently amplified.

Gene Isolation

Gene isolation is often a prerequisite to studying the gene. Gene cloning allows us to isolate genes. This is done by fragmenting the DNA into pieces and checking if a gene or gene fragment is present in the section.

PCR can also be used to isolate genes. If the primers are placed at the boundaries of a gene, the gene would be duplicated much more efficiently than by gene cloning. However, selecting the right boundaries is often difficult. Thus PCR cannot be used to study genes which have not been studied before. In addition, PCR cannot be used on gene longer than 40 kb.

Gene cloning is the only method which can be used with unknown genes or long genes.

Source

Gene Cloning & DNA Analysis by T. A. Brown

Cloning Vectors

To act as cloning vector, a DNA molecule must be capable of entering a host and replicate itself inside the host. In addition, it must ideally be small in size (less than 10 kb) as larger molecules tend to break down during purification and are difficult to manipulate. Two naturally occurring types of DNA molecule satisfy these requirements: Plasmids and Viral Chromosomes.

Plasmids

Plasmids are small circular DNA found in bacteria and some other organisms. Plasmids can replicate independently of the host cell chromosome. Plasmids can carry one ore more genes. Most plasmids possess at least one DNA sequence that can act as an origin of replication, so they are able to multiply within the cell independently of the chromosome.

A few types of plasmids insert themselves into the bacterial chromosome for replication. Such plasmids are called episomes.

The size and copy number of a plasmid is important. Copy number refers to the number of molecules of an individual plasmid that are normally found in a single bacterial cell. Plasmids range from 1 kb to 250 kb. Plasmid smaller than 10 kb are preferred for vectors.

Plasmids can be conjugative or non-conjugative. Conjugative plasmids are characterized by their ability to promote sexual conjugation between bacterial cells. Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact. Conjugation and plasmid transfer are controlled by a set of transfer or tra genes which are present in conjugative genes but absent in non-conjugative genes.

To coexist in a cell, different plasmids must be compatible. If two plasmids are incompatible, one of them would quickly be lost from the cell. Thus plasmid can be assigned to different incompatibility groups. Naturally occuring plasmids are classified on the basis of the main characteristic code by the plasmid genes.

  1. Fertility or F plasmids: carry only tra genes and have no characteristic beyond the ability to promote conjugation
  2. Resistance or R plasmids: carry antibiotic resistance genes. Used as selectors in the lab
  3. Col plasmids: code for colicins which are proteins that kill other bacteria
  4. Degradative plasmids: allows the host bacteria to metabolize unusual molecules
  5. Virulence plasmids: confer pathogenicity on the host bacteria

Bacteriophages

Bacteriophages or phages are viruses that specifically infect bacteria. Viral chromosomes and chromosomes of bacteriophages, in particular, are ideal for inserting DNA into the host chromosome where they are replicated by the host chromosome.

Phages are very simple in structure. They consist DNA surrounded by a protective coat or capsid made up of proteins.

The general pattern of infection is a three-step process:

1. The phage attaches to the cell membrane of a bacteria and injects its DNA into the cell.
2. The inserted DNA is replicated by the bacteria's cell machinery
3. Protein components of the phage are assembled and the phages are released from the host

Lytic Cycle
If the cell infection cycle is very rapid (completed within minutes), it is called a lytic cycle as the release of phage particles is associated with lysis of bacterial cell. DNA is replicated immediately after insertion followed by capsid synthesis. The phage DNA is never maintained in a stable condition in the host cell.

Lysogenic Phages

Lysogenic or temperate phages are those that can either multiply via the lytic cycle or enter a quiescent state in the cell. In this quiescent state most of the phage genes are not transcribed; the phage genome exists in a repressed state. The phage DNA in this repressed state is called a prophage because it is not a phage but it has the potential to produce phage. In most cases the phage DNA actually integrates into the host chromosome and is replicated along with the host chromosome and passed on to the daughter cells. The cell harboring a prophage is not adversely affected by the presence of the prophage and the lysogenic state may persist indefinitely. The cell harboring a prophage is termed a lysogen. [3]

λ phage is a typical lysogenic phage. In an M13 phage, new phage particles are continuously assembled and released from the cell. The phage DNA is neither quiescent for integrated in the bacterial chromosome. Cell lysis never occurs. Both λ and M13 have found major roles as cloning vectors.

Properties of λ DNA
In λ DNA, genes coding for functionally related proteins (such as capsid proteins) are clustered together. The linear molecule has a 12 nucleotide single strand stretch which is complementary to the other end. Thus the molecule can become circular. Such complementary ends are called cohesive ends or cos sites. Cos sites allows the DNA to be inserted into the bacterial genome, circularization is a necessary prerequisite. Cos sites also act as recognition sequences for endonucleases that cleave DNA at cos site, producing individual λ genomes.

M13
M13 is filamentous has a completely different structure from λ. M13 genome is much smaller than λ genome, circular and single stranded. M13 DNA is inserted into E. coli during sexual conjugation via a pilus. Once inside the DNA is complemented and replicated. The DNA is never inserted in the bacterial genome.

The small size of M13 genome is very desirable. The double-stranded replicative form (RF) of M13 genome behaves like a plasmid and can be treated as such for experimental purposes. It is easily prepared and reintroduced in the cell by transfection. Using M13 vector is an easy and reliable way of obtaining single-stranded DNA.

Sources

[1] Gene cloning and DNA analysis by T. A. Brown
[2] http://en.wikipedia.org/wiki/Bacterial_conjugation
[3] http://pathmicro.med.sc.edu/mayer/phage.htm

Introducing vectors into cells

Cloning serves two main purposes. First to allow a large number of recombinant DNA molecules to be produced from a limited amount of starting material. Second important function is purification. Ligation mixture usually contains:

1. Unligated vector molecules
2. Unligated DNA fragments
3. Vector molecules that have been recircularized without new DNA being inserted
4. Recombinant DNA molecules that carry the wrong inserted DNA fragment

Unligated vector molecules and DNA vectors are usually degraded by bacterial enzymes. Self ligated and incorrect recombinant plasmids replicate just as efficiently as desired molecules. The challenge is therefore to identify the colonies that contain the correct recombinant plasmids.

Transformation - uptake of DNA by bacterial cells

Most species of bacteria are able to take up DNA molecules from the medium they grow in. Often DNA molecules are degraded but occasionally they are able to survive and replicate in the host cell. In particular, this happens if the DNA molecule is a plasmid with an origin of replication recognized by host. Most species of bacteria are not efficient at uptaking DNA.

To induce transformation, we place the bacteria in ice cold salt solution. Then we briefly raise the temperature to 42 degrees. This cell is now called competent.

Cells that contain the plasmid pBR322 are resistant to antibiotics. This is used for selection. It allows us to distinguish between transformants and non-transformants.

Insertional Inactivation
Harder problem to solve is to determine which of the transformed colonies comprise cells that contain recombinant DNA molecules, and which contain self-ligated vector molecules. Insertional inactivation is the inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. Recombinants can therefore be identified because the characteristic coded by the inactivated gene is no longer visible.

pBR322 contains genes which code for ampicillin resistance and tetracycline resistance. BamHI cuts in the middle of the gene which codes for tetracycline resistance. If a gene is inserted here, the plasmid loses it ability to code for tetracycline resistance. Thus the plasmid containing the recombinant gene is resistant to ampicillin but sensitive to tetracycline. To screen, we use replica plates.

The pUC8 plasmid is ampicillin resistant and contains a gene lac Z' which partially codes for β galactosidase. To make the plasmid capable of coding for the whole protein, we add the missing DNA along with the recombinant gene. The host which contains the plasmid pUC8 is resistant to ampicillin and is also capable of producing β galactosidase.

Eukaryotic Transfection

This equivalent to transformation, with the difference being that phage DNA rather than a plasmid is involved. Just as with a plasmid, the purified phage DNA or recombinant phage molecule is mixed with competent E. coli cells and DNA uptake is induced by heat shock. Transfection is the standard method for introducing double-stranded RF form of an M13 cloning vector into E. coli.

Prokaryotic Transduction - in vitro packaging of λ cloning vectors

Transfection of λ phages is not a very efficient system when compared to infecting the bacteria with recombinant λ phages. The idea is to first create recombinant λ phages and then to infect the bacteria with them.

Source

Gene Cloning & DNA Analysis by T. A. Brown

Manipulating DNA

Purifying DNA from Living Cells

Genetic engineers require 3 types of DNA. The total cell DNA, plasmid DNA, and phage DNA. M13 is the exception, where plasmids rather than phage techniques are used.

To extract DNA, we need to follow the following steps:

1. Harvest bacteria/viruses
2. Cell is broken to release their contents
3. Cell extract is treated to remove all components except the DNA
4. The resulting DNA solution is concentrated

Manipulating DNA with enzymes

Once pure sample are prepared, we need to construct recombinant DNA molecules. To prepare recombinant DNA, vector DNA is cut at specific places, desired DNA is inserted at then the DNA is joined. Cutting and joining are done by restriction enzymes. DNA manipulative enzymes can be grouped into 5 classes depending on the type of reaction they catalyze:

1. Nucleases: are enzymes that cut, shorten, or degrade nucleic acid molecules
a. Exonucleases: remove nucleotides one at a time from the end of a DNA molecule - Exonuclease III
b. Endonucleases: are able to break internal phosphodiester bonds within a DNA molecule - Bal31
2. Ligases: join nucleic acid molecules together
3. Polymerases: make copies of molecules
4. Modifying enzymes: remove or add chemical groups
5. Topoisomerases: introduce or remove supercoils from covalently closed circular DNA - not used in genetic engineering

Polymerases
DNA polymerases are enzymes that synthesize a new strand of DNA complementary to an existing DNA or RNA template. There are 4 types of polymerases used in genetic engineering:

1. Basic reaction: a new DNA strand is synthesized 5' to 3'
2. DNA polymerase I: fills in the nicks (open spaces) and continues replacing existing nucleotides - Taq DNA polymerase
3. Klenow Fragment: only fills in the nicks
4. Reverse transcriptase: uses a template of RNA to create DNA. Useful in cDNA techniques

Type II restriction endonuclease
Each type II restriction endonuclease cuts DNA and specific nucleotide sequences and nowhere else. This is why these enzymes are very important in gene cloning. Some make blunt cuts while others make sticky (cohesive) ends. Examples: EcoRI, BamHI, HindIII, etc.

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