Reminder: an excellent (though slightly out of date) web site to visit for relevant information is The US Department of Energy's Primer of Molecular Genetics. I have reproduced one or two figures from that primer here.
This is the general term given to the isolation of a specific fragment of target (e.g. human) DNA by propagating that fragment in a microorganism (usually a bacterium but sometimes a yeast). Using standard microbiological techniques, a single microorganism can be isolated and grown in culture carrying within its genome one small fragment of human DNA. The human DNA can readily be purified from the bacterium in gram quantities if necessary. For a more detailed explanation start here
All clones come from libraries. Essentially these may be only one or other of cDNA or genomic, derived respectively from mRNA or genomic DNA, and it is vital to remember the distinction between them.
Southern and Northern Blotting
Before PCR and cheap fast sequencing changed our view of the universe that is genetics, the Southern Blot was a universal workhorse. There was not an experiment in molecular genetics which did not at some stage employ a Southern Blot. It is still a useful tool and you need to know about it so that you can interpret historical data.
Named after its inventor, Prof. Ed. Southern, the blot is a fast way of analysing a small number of DNA fragments which may be present in a complex mixture. For instance, suppose that we wish to ask whether the sickle cell mutation is present in an individual and the only material which we have available is a DNA sample. We could employ a Southern Blot:
- DNA is first digested with a restriction endonuclease and then separated according to the size of DNA fragments by electrophoresis through an agarose gel. Small fragments are able to migrate more quickly through the gel than are long fragments. Most restriction enzymes will digest human DNA into about a million fragments. We will want to compare only those which contain the human beta globin gene. To do this the DNA is first denatured, (made single stranded), by treatment with NaOH. It is then transfered to the surface of a nylon filter by blotting as in part a) of the figure.
- A probe is made. Usually this means incorporating a label into a fragment of DNA from the target sequence (beta globin in this case). The label may be either a radioactive isotope (often 32P) or a chemical hapten such as biotin attached via a long side chain.
- The single stranded probe is annealed to its target sequence which is then stringently washed to remove any probe which has bound other than by perfect fornation of a long run of complementary hydrogen bonds. See section b) of the figure.
- Finally the position of the annealed probe is determined, (by autoradiography in the case of a radioactive probe), see c).
In the case of the sickle cell mutation, the single base change involved, as well as causing a missense mutation in the beta globin gene, also causes the disappearance of a restriction site for the enzyme MstII. As a consequence the size of the restriction fragment containing the 5´ end of the gene is altered from 1.15kb to 1.35kb. See the figure below where MstII sites are shown as arrows.
The Polymerase Chain Reaction
This invention has revolutionised molecular genetics by doing away with the need to clone DNA in many circumstances where it used to be necessary. It is so poweful that it has made it possible to produce microgram amounts (that's a lot!) of DNA starting from just a single molecule. This has applications in forensic science, in archeology (Neandertal mitochondrial DNA amplified from ancient bones by PCR was recently sequenced), and in medicine where, for example, it can enable antenatal DNA tests to be performed in just a few hours work and large populations can be screened for particular mutations very quickly and cheaply.
At the heart of the so called "new genetics" is our ability to sequence DNA rapidly and cheaply. With knowledge of sequence comes knowledge of gene structure and very often a beginning of understanding of gene function. The simplest method of sequencing DNA was invented by Dr. Fred Sanger for which he was awarded his second Nobel Prize. In the UK our major national Human Genome Project DNA sequencing centre is named after him, the Sanger Centre in Cambridgeshire. You can visit its home page here if you wish.
The Sanger method is also known as the "dideoxy chain termination method".
- The DNA to be sequenced must be obtained in pure form, either by cloning or by PCR.
- The DNA strands must be separated (usually by cloning into a vector, M13 bacteriophage, which has a single stranded phase to its life cycle but sometimes by some other cunning technique or even just by heating).
- A primer is allowed to anneal to known sequence at one end of the target sequence. The known sequence may be part of the bacteriophage DNA flanking the cloned DNA.
- Deoxynucleotide triphosphates (dATP, dCTP, dGTP and dTTP) and DNA polymerase are added and the primer sequence is thus elongated along the target "template" DNA.
- Also included in the mixture are small amounts of the four dideoxynucleotide triphosphates (ddNTPs) each tagged with a different coloured fluorescent dye. When a molecule of ddNTP is incorporated into an elongating DNA strand it prevents further chain elongation because it lacks the necessary terminal 3´ OH group.
- The products of synthesis are then separated according to size by electrophoresis on a polyacrylamide gel. This figure shows an earlier method in which the four bases were read in separate reactions and a radiocative "label" was incorporated into the newly synthesised DNA. But the principle is the same.
- As the products pass a point on the gel one by one, the coloured dye tags can be read by a laser scanner and computer.
STSs and ESTs
PCR and sequencing together have made possible the creation of useful landmarks in the genome. These are several thousand short fragments of known DNA sequence whose presence in any DNA sample can be tested by PCR. They are known as STSs (Sequence Tagged Sites). If an STS is part of a transcribed sequence it is known as an EST (Expressed Sequence Tag). Hundreds of thousands of ESTs have been created and can be accessed by computer. One major attempt to classify them all is known as Unigene
The topics include:
- gene cloning
- Southern and Northern blotting
- The Polymerase Chain Reaction
- DNA sequencing
- Mange and Mange Chapters 6 and 8 (good chapters which cover the essentials and more)
- Lewis is weak on methodology but strong on applications. Partial information is in Chapters 8 and 17 (pp316 - 322 (the rest of the chapter is interesting but not directly relevant)), you could also read chapter 21
- Mueller and Young Chapter 4
- Jorde et al. Chapter 3 (pp 42 - 56)
- Thompson McInnes and Willard Chapter 5 (an excellent account)
- Connor and Ferguson-Smith Chapter 3 (too little (and too advanced?) - but have a look if its all that's available)
- If you wanted to study the stucture and regulation of the chromosome 14 linked gene coding for alpha-1-antitrypsin which is expressed strongly in liver but weakly or not at all elsewhere in the body, which of the following resources might provide useful material?
- A human genomic library in a cosmid vector made from leukocyte DNA
- A cDNA library made from cardiac muscle
- A cDNA library made from foetal liver
- A cDNA library made from adult liver
- An arrayed human genomic library in a cosmid vector made from flow sorted chromosomes 9
- A YAC clone containing STSs known to flank the alpha-1-antitrypsin gene on chromosome 14
- How might you save yourself a great deal of unnecessary trouble by finding publically available clones to aid the study in the previous question?
- The genes which when mutant cause the disease Tuberous sclerosis have finally been isolated after a long search. This could not have been accomplished without a very great deal of help from the families in which the disease is present. Apart from their natural curiosity into the nature of the disease which was afflicting them, what was in it for them? In other words, how does finding the genes help the families?