DNA Sequencing

Contents

DNA Extraction

DNA is a long and thin macromolecule with length up to 2 meters but only 10-7 cm in width. It is invisible to the naked eyes. However, the atomic force microscope (AFM) has been used since the mid 1980s to produce topographic maps of nanostructures including DNA. In atomic force microscopy, an extremely sharp tip senses the atomic shape of a sample while a computer records the path of the tip and slowly builds up a three-dimensional image. One of the first images of DNA is shown in Figure 01, where the white blobs are repairing proteins bound to the DNA. There is a crude method to extract DNA from the cells. The result doesn't show the individual macromolecule but appears in the form of cloudy substance. The steps, which are applicable at home and in school, are outlined in the following:

Figure 01 DNA Image [view large image]

	Figure 02 displays all the materials and equipments to carry out the experiment. They are items commonly found in the household. Pour 3/8 cup of water into a cup. Add 1/4 teaspoon of salt and clear liquid soap into the cup, and stir for the salt to dissolve. The soap causes the cell walls to break up, freeing the DNA inside. The salt is for keeping the DNA molecules together.
Figure 02 The Prop [view large image]	Place the organic materials into the sandwich bag or blender and mash or crush until completely pulverized. This is to break the cells apart. Add 2 teaspoons of the soap-salt solution to the bag. Mix gently by compressing or rocking the bag for at least 1 minute.

Pour the mixture through coffee filter into a clear jar. Let it drain into the jar for 10 minutes. Meanwhile, pour 1/4 coup of rubbing alcohol into another clear jar and put the jar in the freezer or a bowl of ice. The alcohol helps strip the water molecules from the outside of the DNA molecules, causing the molecule to collapse on itself and separate from the proteins in the solution. After 10 minutes have elapsed, pour the strained liquid into the alcohol. Let the jar sit still for at least 5 minutes. The final product contains a layer of proteins and a cloudy layer of DNA clumped together as shown in Figure 03. The naked eyes cannot resolve the individual macromolecules within. Occasionally the presence of

Figure 03 DNA Extraction [view large image]

the DNA molecule is betrayed by a string of tiny air bubbles suspending in the middle of the solution.

Releasing the DNA from the cell is only the initial step in "professional extraction kit". Figure 04 shows the many steps in one of the products in the market.

	1. Add tissue sample into the preparation solution to break the cells apart. 2. Use the extraction solution to break up the cell walls. 3. Add neutralization solution to remove impurities, which inhibit the following actions. 4. Mix primers in the solution to prepare for DNA copying (amplification).
Figure 04 Extraction Kit [view large image]	5. Run the cloning process called PCR (Polymerase Chain Reaction). 6. Load the end product to the gel for DNA analysis.

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Gel Electrophoresis

The gel electrophoresis method was developed in the late 1960's. It is a fundamental tool for DNA sequencing. The following outlines steps for its construction (see Figure 05):



1. Agarose powder is mixed with electrophoresis buffer (for establishing pH level, and providing ions to support conductivity, usually Tris-acetate-EDTA (TAE) or Tris-borate-EDTA (TBE)) to the desired concentration, then heated in a microwave oven until completely melted. Most commonly, ethidium bromide (a fluorescent dye that intercalates between bases of nucleic acids and allows very convenient detection of DNA fragments in gels) is added to the gel at this point. After cooling the solution to about 60C, it is poured into a casting tray containing a sample comb and allowed to solidify at room temperature. The insert in Figure 05

Figure 05 Gel Electrophoresis [view large image]

shows the effect of agarose concentration on the migration of DNA with 7% resolves longer piece better while the 1.5% is good for the shorter one. 2. After the gel has solidified, the comb is removed to form wells for the samples.

The gel, still in its plastic tray, is inserted horizontally or vertically (depending on the design of the apparatus) into the electrophoresis chamber and covered with buffer. Samples containing DNA mixed with loading buffer (which contains something dense, e.g., glycerol to allow the sample to "fall" into the sample wells, and one or two tracking dyes to allow visual monitoring) are then pipeted into the sample wells, the lid and power leads are placed on the apparatus, and a voltage of no more than 5 volts per cm is applied (the cm value is the distance between the two electrodes).
3. In the electrophoresis buffer the DNA dissociates into a negatively charged moiety and a hydrogen ion. Thus the DNA anion will migrate toward the positive electrode. Since the DNA molecules have to wiggle through the microscopic meshes within the gel (called viscosity in physics), the rate of migration depends on their length - the shorter one moves faster, i.e., the mobility is inversely proportional to the log₁₀ of the molecular weight. Now the DNA fragments of different size have been separated into groups as shown in Figure 05. This is an important step toward DNA sequencing, but the identity of the DNA is still unknown.

The great length of DNA molecules was the main stumbling block that prevented its sequencing. The problem was resolved by the discovery that bacteria use an enzyme to restrict infection by certain bacteriophages - hence the term restriction enzyme. The restriction enzymes are since used to cut whole piece of DNA into fragments (Figure 06a). Specifically, there are enzymes to cut DNA precisely after an adenosine nucleotide (A) or T, or G, or C.

		The Maxam-Gilbert method of DNA sequencing separates the DNA sample into four groups each one treated with a specific restriction enzyme for A, T, G, or C. After this, all four groups are placed in the same apparatus for gel electrophoresis. The resulting DNA sequence is shown in Figure 06a. The process can be automated by attaching different fluorescent dye to the end of the DNA fragments in each group and
Figure 06a Restriction Enzyme [view large image]	Figure 06b Automation [view large image]	read off by a scanning detector hooking up to a computer (Figure 06b).

This technique can be used to sequence fragments of DNA up to many hundreds of units. It has since been refined to perfection by the Sanger's method.

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PCR (Polymerase Chain Reaction)

PCR is a process of DNA amplification (replication). Polymerase is an enzyme responsible for DNA synthesis, while "Chain Reaction" means exponential growth (at the rate of 2ⁿ where n denotes the number of generation) - hence the PCR name. The process is essential for sequencing unique piece of DNA such as those from the genomic specimen, the fossil, or a single hair in a crime scene. Here's a summary of each step in running the PCR (see Figure 07):

1. A target sequence is chosen on the DNA. The sequence had to be known, or at least its two termini.
2. Since DNA replication always runs from the 5' end to the 3' end, two DNA primers were synthesized for each strand. The primer is a small complementary to the 3' end of the original DNA about 20 bp long, it acts as an anchor

point for DNA polymerase and as initiator of the copying process. 3. Modern technique uses the Taq polymerase (from a microbe in hot springs) to add nucleotide to the new strand. It has the advantage of tolerance to heating up to 94oC - well beyond the 72oC for the extension phase. 4. All four DNA nucleotide building blocks are added in sufficient quantity. 5. The sample is heated to a temperature of up to 98oC to separate the complementary strands. This step is called denaturation. 6. Then the sample is cooled. During the cooling stage, the synthetic primers found complementary sites on the separated DNA strands, whereas the two long DNA strands were unable to find each other because they were present in minute concentration. This process is called primer annealing. 7. The polymerase extended the two primers in opposite directions. As a result, two daughter DNA appeared. 8. Initially, the new DNA carries long single-stranded tail. Only at the 3rd cycle

Figure 07 PCR Process [view large image]

of denaturation-annealing-extension do first authentic copy of the molecules appear. The process can make billion copies by the 30th cycle.

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DNA Sequencing

Frederick Sanger is among the rare breed of individuals, who won the Nobel Prize twice - once in 1958 for identifying amino acid sequences in proteins and the second time in 1980 for DNA sequencing. The following summarizes his contribution to the method carrying his name (see Figure 08a):



1. Primer together with DNA polymerase and nucleotides are added to the sample. The steps proceed similarly to the PCR process up to the extension phase. 2. Just as in the Maxam-Gilbert method, the sample is divided into four groups. 3. The novalty of this method involves molecules called dideoxynucleoside triphosphates (ddNTPs), which are similar to the normal nucleotides except that the oxygen atom is missing in the 3' spot of the deoxyribose. They are designated as ddTTP, ddATP, ddCTP, and ddGTP corresponding respectively to T, A, C, and G. The absence of the oxygen atom terminates the primer extension reaction when the normal nucleotides are replaced by the ddNTPs. In addition, each of the ddNTPs emits a different fluorescent signal to identify the T, A, C, G bases. 4. The sample is processed by gel electrophoresis as usual revealing the DNA complementary sequence by the end label of each fragment as shown in Figure 08a.

Figure 08a DNA Sequencing [view large image]

Current methods can directly sequence DNA fragments 300-1000 nucleotides long in a single reaction. [3]. The main obstacle to sequencing DNA fragments above this size limit is insufficient power of separation for resolving large DNA fragments that differ in length by only one nucleotide.

As companies in the DNA sequencing market jostling to be the first, the fastest and the cheapest to sequence large amounts of DNA, a technique has been developed in 2008 to read individual letters from single molecules of DNA skipping the need for the amplifying (copying) step, which can introduce errors and does not work well for some DNA fragments, making it difficult to sequence a genome’s full complement of DNA. By avoiding these complications, the single-molecule approaches may reduce the time and the cost of sequencing an individual’s genome. The new method begins by chopping the genome into small fragments. An enzyme then attaches a short DNA tag onto the end of each fragment. The tag binds to a complementary DNA molecule attached to a platform, anchoring the DNA fragment in place. A DNA replicating enzyme is then added along with a stock of DNA 'letters' or bases carrying fluorescent labels. As the fluorescent DNA forms into strands that complement the genome fragments, a camera takes a picture of each newly added base. However, such technique can only identify fragments of about 30 DNA letters. Longer fragments have to be stitched together via overlapping ends to a length over 1,000 bases long. It is said that the human genome sequence can be assembled for less than US$1,000/person in less than five years.

Another single molecule sequencing technique is to keep the concentration of one of the four nucleotides required for transcription extremely low, pausing events will occur as the RNAp attempts to add this nucleotide to the growing chain. Holding each of the four nucleotides at the rate-limiting concentrations in four otherwise iden-tical assays, it shows that the process is able to sequence the underlying DNA up to 32 base pairs by analyzing the ordered sequence of pausing in each of the 4 cases.

Figure 08b Single DNA Sequencing [view large image]

An example of their transcription data is shown on the top of Figure 08b, which plots the position vs time for each of the nucleotide in low concentration (the hori-zontal step pinpoints the position). The inferred sequence is shown in the bottom.

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Large Scale Sequencing

Large-scale sequencing aims at sequencing very long DNA fragments. Even relatively small bacterial genomes contain millions of nucleotides, and the human chromosome 1 alone contains about 246 million bases. Therefore, some approaches consist of cutting (with restriction enzymes) or shearing (with mechanical forces) large DNA fragments into shorter DNA fragments. The fragmented DNA is cloned into a DNA vector, usually a bacterial plasmid, and amplified in Escherichia coli as

Figure 09 Large Scale Sequencing [view large image]

the bacteria divides. These steps are shown in Figure 09.

The first method of sequencing a genome (the Hierarchical Shotgun Sequencing Method) , employed by the publicly funded Human Genome Project, involves the following steps (Figure 10) :



1. The source DNA is mapped with some known genetic markers (landmark). 2. It is cut into smaller pieces called sub-clones according to the genetic markers. This allows the sub-clones to be placed in an order based on the structure of the DNA. 3. The sub-clones are further divided into fragments. 4. The fragments are then sequenced and matched at the overlaps. 5. The sequenced sub-clones are linked up (according to the order) to produce the DNA contig, which is the de-coded version of the original source DNA. As this method progresses, larger and larger contigs will be produced, until a single ordered contig of the genome is achieved.

Figure 10 Hierarchical Shotgun [view large image]

This method saves labour by allowing sequencing of each individual clone in different laboratories and that each stretch of DNA only needs to be sequenced once. The disadvantage is the slow process of sub-cloning and mapping of the source clones, requiring significant human manipulation.


A second method is the "Shotgun Sequencing" (Figure 11), which was used by the privately funded company Celera Genomics to sequence the human genome. This method was the subject of a good deal of debate, as it is relatively crude and prone to error in comparison to the method employed by the Human Genome Project. It involves each piece of DNA being cut into smaller fragments in the same way as shown in Figure 09. There is no physical genetic map to

order the fragments. Instead, each fragment is sequenced first, and then overlapping sequences are joined together to create the contig. In other words, random fragments are sequenced (as they are not ordered) in the hopes that overlapping sequences will be found to piece together the contiguous sequence. To ensure that that the whole source clone has been sequenced, this stretch of DNA must be sequenced numerous times (represented by the multiple rows of fragments in Figure 11) to produce an ordered overlapping sequence. Gaps in this process will occur where no fragment is found. This method is advantageous in that the laborious process of mapping and sub-cloning is eliminated. So, while it requires much more sequencing compared to the Hierarchical Shotgun, it proves to be much more economical and faster due to the sequencing reactions being virtually fully automated and the sequences being assembled by computer programs.

Figure 11 Shotgun Sequencing [view large image]

The best automated-gene sequencers claim output of up to 7200 bases per hour. A new technique, sequencing-by-hybridization (pairing) promises to sequence more than four times faster. The steps are summarized below (Figure 12):



1. Single strand DNA fragments (of unknown identity) with fluorescent tags are poured into the apparatus. 2. Short DNA fragments of known sequence are already immobilized on the microchip (about 1cm x 1 cm). The known sequence in the example consists of 6 bases each resulting in 46 = 4096 different combinations. The known sequence are arranged in a vertical column contrary to the illustration in the diagram. 3. The fluorescent labels reveal 6 complementary bases of the unknown sequence as it is aligned with one of the column.

Figure 12 Hybridization [view large image]

4. The entire sequence can be derived from the overlaps. 5. The unknown is just the complementary of the above.

Figure 13 shows the schematic of the micro array marketed by Affymetrix. There are 6.5 million locations (columns) on the array with millions of identical DNA strands in each location, and each strand consists of 25 bases (called probes).

		The array is rinsed and washed with a fluorescent stain that clings to the biotin on the strands of the human sample that remain. A laser causes them to glow, and the DNA is analyzed based on which probes on the array they matched with. The latest offer by Roche claims to capture up to 5 Mb of
Figure 13 Affymetrix Micro Array [view large image]	Figure 14 Roche Micro Array [view large image]	total sequence on a single array (see Figure 14).