|
|
|
|
DNA
Sequencing - Sequencing DNA refers to the order of nucleotide bases along its sugar phosphate backbone.
Through the specificity of base pairing a double helix of DNA maintains a constant structure irrespective of its particular sequence.
The difference between individual molecules of DNA lies in their particular sequences of base pairs, not in gross changes of structure.
DNA exists in the form of very long chains (5,000 base pairs long in one bacteriophage; 240,000,000 bp long in the human chromosome).
|
No techniques can determine the sequence of bases in an entire chromosome in a single experiment; so it is necessary to cut the chromosomes into fragments of manageable size (a few hundred base pairs long) and purify each fragment type.
This is done by cloning the fragment into a plasmid or viral DNA vector. After amplification, the cloned DNA segments are released for sequencing by cleavage with a restriction endonuclease.
At one time sequencing nucleic acids was extremely difficult; even if enough material could be obtained in a pure state, the actual sequencing process was prohibitive.
|
Some molecules, such as tRNAs, were sequenced but the technique was not widely employed. Protein sequencing was time consuming but could be achieved if the protein was of sufficient purity. Protein purification is still a problem but pure DNA sequences can be obtained in large quantities by molecular cloning.
Firstly, we must generate a series of single stranded DNA molecules, each molecule one base longer than the last. DNA molecules of the same sequence but differing in length by as little as one base at one end can be separated by electrophoresis on acrylamide gels.
|
|
This extraordinary sensitivity to size is made use of in the base-sequencing procedure. Two approaches have been used to obtain these sets of bands. One is to use chemical reactions that cleave DNA at individual bases. The other is to use an enzymatic reaction in which DNA is synthesized in vitro in such a way that the reaction terminates specifically at the position corresponding to a given base.
To determine the sequence of the molecule by either approach, it is subjected to the appropriate protocol in four separate reactions, each reaction specific for one of the four bases, whereby a cut is made in the DNA next to a G, an A, any pyrimidine (that is T or C), and a C. DNA sequencing was revolutionized by Maxam and Gilbert (1977,1980) (Mantell, Matthews, and McKee, 1,985) and their method relies on our ability to manipulate DNA with a variety of enzymes.
|
|
|
A restriction fragment of DNA is labeled at either its 5- or 3-end with (32p), using either polynucleotide kinase or terminal transferase. An enzyme is selected from a restriction map, which will remove a small piece from one end of the molecule, leaving just one end labeled.
The DNA is then chemically cloven at specific residues in five different reactions, but the reactions only partially completed.
The partial digestion products are separated on a polyacrylamide gel and autoradiographed.
Only fragments containing the labeled terminus are visualized; the sequence can then be deduced from the order of fragments from the different digestions.
|
A different method was described by Sanger et al. (1977), known a$ the dideoxy or chain terminator method. Again, a series of radiolabeled fragments of increasing size are generated but this time by copying single stranded DNA in the presence of a labeled nucleotide, with DNA polymerase.
The polymerase will also incorporate dideoxy nucleotide into the strand, but once incorporated there is no free hydroxyl to accept the next residue and the chain is therefore terminated.
By using a carefully selected ratio of dideoxy nucleotide to its deoxy nucleotide, the terminator is put in randomly, terminating different chains at many different points. The fragments are analyzed in essentially the same way as those from Maxam and Gilbert sequencing.
This method requires a primer to be used in order to initiate DNA synthesis. Generally the DNA to be sequenced is cloned into a phage known as M13, which has been carefully manipulated to allow a universal primer to initiate sequencing (Messing et al., 1981).
Sequence analysis of cloned DNA has yielded a large amount of information, particularly about sequences at the beginning and end of genes involved in the possible control of the expression of the gene. Sequencing is a powerful tool and provides by far the most detailed analysis of a particular piece of DNA.
The technology does rely on information obtained by using other techniques, however, restriction maps for example, and cannot stand alone. The information obtained must also be coupled with other investigations before interpretation of the function of any specific sequences can be made.
First, a radioactive 32p atom is chemically added to the two 5ends of each fragment and then the complementary single strands separated and purified.
Each complementary strand is sequenced independently and the sequences then compared for confirmation. A sample containing only one of the complementary strands is subjected to four different protocols, as mentioned above.
All four parts are then electrophoresed and the bands are located by placing autoradiographic film (a film that responds to radioactivity) on the gel. (Note that when a 5'- 32P-lableled molecule is cloven, only one of the two fragments produced contains 32p and only that one is detected) The positions of A and G in the single strand are determined by the following rules:
1.If a fragment containing n nucleotides is generated by a chemical treatment that causes cleavage at the site of a particular base, then that base is present in position n +1 of the DNA strand, the position being counted from the 5' -ends.
2. If a band containing n nucleotides is present in the A, G, C or only in T + C parts, then an A, G, C, or T respectively exists at position n + 1 in the original molecule.
All four samples are electrophoresed simultaneously, enabling all bands to be seen in a single gel. The sequence is read directly from the gel. The shortest fragments are those that move the fastest and farthest. Each fragment contains the original 5'_32p group; the sequence can therefore be read from the bottom to the top of the gel. In the chemical degradation approach, a DNA restriction fragment is first radiolabeled at its 5- or 3 -end.
The DNA is partially degraded and modified into, a set of base-derivative bases, and cleavage of the DNA molecule is done by the addition of chemicals. In the enzymatic approach an oligonucleotide primer is hybridized to a singles) rand DNA template adjacent to the target region to be sequenced.
| |
