Sequencing is the method to get the order of DNA basepairs of a DNA fragment. This fragment can be small (like 500 bp) or a whole genome of an organism. One of the major methods of DNA sequencing in known as chain termination sequencing, dideoxy sequencing, or Sanger sequencing after its inventor biochemist Frederick Sanger. The method is elegantly simple. While DNA chains are normally made up of deoxynucleotides (dNTPs), the Sanger method uses also dideoxynucleotides.
Dideoxynucleotides (ddNTPs) are missing a hydroxy (OH) group at the 3' position. This position is normally where one nucleotide attaches to another to form a chain. If there is no OH group in the 3' position, the additional nucleotides cannot be added to the chain, thus interrupting chain elongation. A small fraction of one of the bases will contain stopnucleotides. This means that everytime that nucleotide is added, a fraction of the strings will stop growing and keep the length it has reached at that time. When you first devide your sample in four tubes, you can do this procedure four times. This means with all the four basepairs. When you run these four samples on a gel, you can read the sequence from the smallest fragment to the largest..
Since 1986 the process of reading the sequence can be done with an automated fluorescence sequencer. The automated sequencer runs on the same principle as the Sanger method (dideoxynucleotide chain termination). But here a laser constantly scans the bottom of the gel, detecting the bands that move down the gel. Where the manual method uses radioactive labeling, automated cappilary sequencing uses fluorescent tags on the ddNTPs (a different dye for each nucleotide). This makes it possible for all four reactions (dGTP, dATP, dCTP, and dTTP) to be run in one lane and increases the speed of the process four times. The runs are fully automated nowadays, and the gels are replaced by cappilaries. This is a very efficient method and is very useful for fast and automatically sequencing of large DNA fragments.
More about sequencing on the history page.
There are two methods of deviding the genome in smaller parts for large-scale sequencing:
- The Conventional Method
- Shotgunning
The Conventional Method
Once scientists use PCR to create many copies of a single strand of the DNA fragment they begin to synthesis the location of each letter. The original method involves the following:
- Step 1: Place identical DNA strands into four test tubes, each one containing a ddNTP that resembles one of the four nucleotides in DNA (A, T, C, G) and lots of dNTPs (which are free-floating nucleotides) that also resemble the DNA letter, except they do not build functioning DNA chains.
- Step 2: Then add the polymerase enzyme and a known primer DNA, similar to the one used in PCR, to the test tubes. The primer marks the beginning of each sequenced string of DNA. In each test tube, the dNTPs, which act like letters in a DNA bond with the complementary nucleotide, thereby copying the original strand. However, the ddNTP in each test tube also bond with the DNA fragments at a probable ratio of 1 bond 100 times it could bond. Each time this happens the copy terminates, thereby creating millions of DNA strings of differing length that start with the same primer and each ending with the same ddNTP nucleotide. This is determined by which of the four test tubes being analysed, since only one of the ddNTPs are in each test tube.
- Step 3: Then use Gel Electrophoresis to arrange the DNA pieces from largest to smallest and X-ray detection to determine the length of the strings.
This is the method that was first developed. Fortunately, contemporary institutes no longer use this exact method but one that is four times faster. By using ddNTPs tagged with fluorescents they no longer need four test tubes, but a single one, which contains all four fluorescent ddNTPs. They then rely on computers to detect the different colours of the ending pieces of the DNA segments after Gel Electrophoresis to determine the letters and length of the DNA chain. More information is given in the History part of this website.
The Shotgun Method
For using the shotgunning method, first A genomic library is made by cutting a whole genome with restriction enzymes and inserting each piece into a bacterium, which is then cloned. These segments are then detected and ordered by computers.
- Step 1: Blend the post PCR DNA string that is to be sequenced into little fragments.
- Step 2: Place the new segments into a test tube filled with the polymerase enzyme and a primer bit of DNA.
- Step 3: Now, like in the original method, let the DNA rebuilds itself with the dNTPs and the fluorescent ddNTPs tagged in the test tubes until all the strings have terminated.
- Step 4: Use Gel Electrophoresis to sort the fragments by size and a computer to record the many DNA fragments lengths. Lastly the computer should process and realign these fragments into the original string, thereby sequencing it.
The advantage of using smaller fragments of the larger DNA chain is that since the time required sequencing the DNA has been greatly shorted. Therefore, machines can sequence the fragments many times in order to achieve a high level of accuracy, by using sequencing software which lines up the DNA by finding overlapping letter sequences in the many pieces after the gel electrophoresis. However, scientists have experienced a few problems when 'shotgunning' DNA strings with many common and reoccurring sequences; therefore researchers using the 'shotgunning' process often sequence the DNA both backwards and forwards to return more accurate results. The following graphic shows how the small DNA fragments are realigned to assemble the full sequence.
Actuality billions of overlapping DNA pieces need to be aligned for an acceptable accuracy. Fortunately, by highly automating the 'shotgunning' method of sequencing, scientists are quickly organising enough DNA pieces to return precise chains faster than competitors using more conventional techniques.
ESTs are also very useful in the mapping of a genome. The 3' ESTs serve as a common source of STSs because of their likelihood of being unique to a particular species and provide the additional feature of pointing directly to an expressed gene. These ESTs gives much information as a reliable genomic landmark for genome mapping.
There are two types of shotgunning:
- Hierarchical (clone by clone)
- Whole genome
Hierarchical shotgunning means, the genome will be broken up into overlapping segments whose relative locations were known; each segment was then shotgun-sequenced. Using the whole genome shotgunning technique, the whole genome will be broken in pieces several times and all pieces are sequenced. Both types detect and order the segments by computer after sequencing each segment. The whole genome shotgunning was invented by Craig Venter's TIGR and the technique was used to sequence several genomes, like the influenza microbe, Drosophila melanogaster and Venter's part of the human genome.
Microarrays that contain all of the approximate 6000 genes of the yeast genome have been available since 1997. The spots are either printed on the microarrays by a robot, or synthesised by photolithography (similarly as in computer chip productions) or by ink-jet printing. The spot diameter is of the order of 0.1 mm, for some microarray types can be even smaller.
