Introduction to Cloning

While I was doing some practice questions, I realised that I was quite shaky on the details of cloning and hybridisation methods, so I'm skipping ahead to this section of the course.

Define a recombinant DNA molecule.

A recombinant DNA molecule is simply a molecule of DNA made up of different fragments that would normally not be found together in nature.

Know the properties of restriction enzymes.

Restriction enzymes, if I remember correctly, originate from bacteria. They cut DNA at specific locations, a function that is used in nature to protect bacteria from bacteriophages (viruses that attack bacteria). To prevent restriction enzymes from cutting the bacteria's own DNA, restriction enzymes work closely with methylases, enzymes that add methyl groups to certain bases in DNA so that the cell can recognise the DNA as its own.

Restriction enzymes only cut the DNA at specific sites. These sites are 4-8 base pairs long and are palindromic- that is, the sequence on the top strand is the same as that on the bottom strand, but reversed (since the strands run antiparallel to each other). When the strands are cut, 3'-OH and 5'-phosphate ends are formed. These ends can be "blunt" ends if, after cutting, the two strands on one side of the break are of even length and the two strands of the other break are also of even length. If this is not the case, then the ends are said to be "sticky" or "cohesive." They are said to have 5' overhangs if the 5' end juts out more, or 3' overhangs if the 3' end juts out more.

Know restriction enzyme-digested DNA with complementary ends can anneal.

After a strand has been cut to leave "sticky" ends, it can anneal to other cut strands that have a complementary sequence on their "sticky" ends. For example, if a strand is cut with a 5' overhang reading AATT, it can bind to an exposed TTAA on another cut strand. DNA ligase then fixes up the backbone.

Know how DNA ligase works.

DNA ligase is the enzyme that "patches up" any gaps in the backbone by covalently linking 3'-OH and 5' phosphate groups. They require ATP to carry out this process.

Know the properties of a plasmid vector and the three properties that make them useful for molecular cloning.

Plasmids are extrachromosomal DNA found in bacteria- that is, plasmids are DNA that aren't considered to be part of the bacteria's chromosomes. They are small, double-stranded and circular. Plasmids contain a multiple cloning site, or polylinker, which has several places where different restriction enzymes can cut. We can use this to insert other DNA into a plasmid for the purposes of cloning, which I will explain in a bit.

Know the steps in inserting a DNA fragment into a plasmid and amplifying it in bacteria.

To insert a DNA fragment in a plasmid, firstly you need to obtain a DNA fragment. You can do this by cutting DNA with a restriction enzyme. You can then cut the plasmid in the polylinker site using the same restriction enzyme. The fragment can then anneal to the plasmid DNA and DNA ligase can then stitch up the backbone.

Cloning plasmids requires a host organism, such as a bacteria. Plasmids can be inserted into bacteria by using heat shock or electroporation (exposure to an electrical field) to make bacterial cells more permeable to DNA. Once this has occurred, the bacteria can divide, producing many copies of the plasmid at the same time.

Be able to describe agarose gel electrophoresis.

Agarose gel electrophoresis is a technique that can be used to determine the length of the fragments generated from cutting the DNA. The length of the fragments can be added up to determine an approximate length of the entire genome.

In agarose gel electrophoresis, the DNA solution is mixed with ethidium bromide, which binds to DNA and RNA and appears fluorescent when viewed under UV light. This solution is then placed in the agarose gel solution. A current is applied to the solution. As DNA is negatively charged, due to its phosphate groups, the DNA migrates through the gel towards the positive electrode. Smaller fragments move more quickly than larger fragments, so at the end of a certain time period the location of the fragments (as seen under UV light) can be compared to fragments of a known length in order to determine the length of the DNA that you are interested in.

Know the characteristics of an expression vector that make it useful for the large scale production of proteins.

This is something that went clean out of my brain, so I revised it just then. Expression vectors are used for large-scale production of proteins. They include a highly active promoter region and a special tag that will be later used for isolating the protein in question. A commonly used tag is a series of bases coding for a cluster of histidine residues (a His tag).

To purify these proteins, affinity chromatography is used. In this technique, extract from the host cell is passed through a column that is filled with metal beads. His tags are more likely to bind to the metal beads, possibly due to the imidazole rings (I'll need to check this- I actually have no idea). As the extract passes through, eventually all that is left are the beads with the His-tagged proteins bound to them. The proteins can then be eluted (i.e. extracted) from the column by reducing the pH of the solvent (again, I'll have to find out why this is so).

Characteristics that would make an expression vector useful would probably be their ability to bind to metal beads or some other adsorbent. Another characteristic would be that they must be quite unique and different to other amino acid sequences commonly found in the cell so as to avoid contamination.