Modern techniques using recombinant DNA allow great insights into the functioning of genes and the molecular pathology of genetic diseases. Often the first step in studying the DNA of an individual involves preparation of genomic DNA. This is a simple procedure in which 20 ml of blood is taken, the lymphocytes are treated to gently break open their cell and nuclear membranes, and chromosomal DNA is chemically extracted. DNA is stable and can be stored frozen for years. Restriction enzymes and gel electrophoresis Genomic DNA can be cut into a number of fragments by enzymes called restriction enzymes, which areobtained from bacteria. Restriction enzymes recognize specific DNA sequences and cut double-stranded DNA at these sites. For example, the enzyme EcoRI will cut DNA wherever it reads the sequence GAATTC, and so human genomic DNA is cut into hundreds of thousands of fragments.
Whenever the genomic DNA from an individual is cut with EcoRI the same ‘restriction fragments’ are produced. As DNA is a negatively charged molecule the genomic DNA that has been digested with a restriction enzyme can be separated according to its size and charge, byelectrophoresing the DNA through a gel matrix. The DNA sample is loaded at one end of the gel, a voltage is applied across the gel and the DNA migrates towards the positive anode. The small fragments move more quickly than the large fragments and so the DNA fragments separate out.
Fragment size can be determined by running fragments of known size on the same gel. Pulsed field gel electrophoresis (PFGE) can be used for separating very long pieces of DNA (hundreds of kilobases) which have been cut by restriction enzymes that cut at rare sites in the genome. In this technique,DNA molecules are subjected to two perpendicular electric fields that are switched on alternately. The DNA molecules are separated on the basis of molecular size and this technique can be used for long-range mapping of the genome to detect major deletions and rearrangements. Southern blotting and DNA probes This technique allows the visualization of individual DNA fragments.
A DNA probe is used to indicate where the fragment of interest lies. DNA probes are useful because a fundamental property of DNA is that when two strands are separated, for example by heating, they will always reassociate and stick together again because of their complementary base sequences. Therefore the presence or position of a particular gene can be identified using a gene ‘probe’ consisting of DNA with a base sequence that is complementary to that of the sequence of interest. A DNA probe is thus a piece of single-stranded DNA that can be labelled with a radioactive isotope or a fluorescent signal. The probe is added to a hybridization solution into which the membrane with the DNA is also placed. The single-stranded probe will locate and bind to its complementary sequence on the blot and can be identified by autoradiography or fluorescence. A similar technique for blotting RNA fragments (which are not cut by restriction enzymes, but which are blotted as full length mRNAs) onto membranes is called Northern blotting and one for blotting proteins is called Western blotting.
The polymerase chain reaction
Minute amounts of DNA can be amplified over a million times within a few hours using this in vitro technique. The exact DNA sequence to be amplified needs o be known because the DNA is amplified between two short (generally 17-25 bp) single-stranded DNA fragments (‘oligonucleotide primers’) which are complementary to the sequences at either end of the DNA of interest. The technique has three steps: the double-stranded genomic DNA is denatured by heat into single-stranded D, A; the reaction is cooled to favour DNA annealing and the primers bind to their target DNA; then a DNA polymerase is used to extend the primers in opposite directions using the target DNA as a template. After one cycle there are two copies of double-stranded DNA; after two cycles there are four copies and this number rises exponentially with the number of cycles. Typically a polymerase chain reaction is set for 25-30 cycles, allowing millions of amplifications. This technique has revolutionized genetic research as minute amounts of DNA not previously amenable to analysis can be amplified, for example from buccal cell scrapings, blood spots, or single embryonic cells.
A particular DNA fragment of interest can be isolated and inserted into a ‘vector’ so that it can be cloned and prepared in large quantities independent of other sequences. Vectors include small circular DNA molecules called plasmids, which are derived from naturally occurring sequences in bacteria; bacteriophages, which are derived from bacterial viruses; or ‘yeast artificial chromosomes’ (YACs), which are derived from DNA sequences found in yeast. Each vector takes an optimum size of DNA insert. Small sequences of a few kilo bases can be inerted into a plasmid. Large sequences of several hundred kilobases can be inserted into a YAC. The DNA fragment of interest is inserted into the vector DNA sequence using an enzyme called a ligase. This takes place in vitro. The next step, cloning, creates many copies of the ‘recombinant DNA molecule’ and takes place in vivo when the plasmid or other vector is placed back into the bacterial (or yeast) host. Bacteria that have successfully taken up the recombinant plasmid can be selected if the plasmid also carries an antibiotic resistance gene (so bacteria without the plasmid die in the presence of antibiotic). The DNA fragment of interest to be cloned may be a restriction fragment. Alternatively it could be DNA (cDNA) which has been copied from an mRNA sequence. cDNA is synthesized by starting with an mR A of interest, which is the template for an enzyme called reverse transcriptase to copy the mRNA into a double-stranded DNA. A cDNA contains all the sequences necessary for a functional gene, only the introns being absent.
Molecular biology, genetic disorders and immunology E. coli cell Vector (with ampillicin resistant gene) These are pools of isolated and cloned DNA sequences that form a permanent resource for further experiments. Two types of library are used: 1 Genomic libraries prepared from genomic DNA that has been digested with restriction enzymes, ligated into a vector and each individual clone has been passed into a bacterial (plasmid, bacteriophage, cosmid) or yeast (YACs) host. A genomic library usually contains almost every sequence in the genome. 2 cDNA libraries prepared from the total mRNA of a tissue, which is copied into cDNA by reverse transcriptase. The cDNA is ligated into a vector and passed into a host as above. A cDNA library should contain sequences derived from all the mRNAs expressed in that tissue type.
A chemical process known as dideoxy sequencing allows the identification of the exact nucleotide sequence of a piece of DNA. The DNA of interest is single stranded and an oligonucleotide primer is annealed adjacent to the region of interest. This primer acts as the starting point for a DNA polymerase to build a new DNA chain complementary to the sequence under investigation. The reaction is carried out in four tubes to each of which a mixture of nucleotides is added, one of which is radioactive. To each tube one dideoxy triphosphate of either adenine,guanine, cytosine or thymine is also added at a low level. These are incorporated into the growing chain and stop enzymatic synthesis (because they lack the necessary 3- hydroxyl group). As the dideoxynucleotides are present at a low concentration, not all the chains in a reaction tube will incorporate a dideoxynucleotide in the same place, so the tubes contain sequences of different lengths but which all terminate with a particular dideoxynucleotide. These fragments are electrophoresed in four columns