DNA Sequencing

DNA Sequencing Definition; DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA. Applications; It is used widely in many fields such as in the field of biotechnology, forensic biology, in diagnostic. A very important accomplishment that was made possible thanks to this technique is revealing the human genome , and as a consequence several genes have been identifyed to associate with certain diseases such as , breast cancer , colorectal cancer, Alzheimer's disease, …….etc. . Ultimately, DNA sequencing will become a part of a patient's medical record, helping physicians to determine the patient's risk of certain diseases and the optimal treatments.

DNA Sequencing A. Manual method B. Automated method Two main methods 1. Sanger dideoxynucleotide chain termination method (Commonly used method) A. Manual method B. Automated method 2. Chemical cleavage method (Maxam and Gilbert method) (Not used nowadays)

Principle of the Sanger Sequencing Method(manual); Classical. Sanger sequencing, published in 1977, relies on base-specific chain terminations in four separate reactions (.A., .G., .C., and .T.) corresponding to the four different nucleotides in the DNA makeup. In the presence of all four deoxynucleotide triphosphates (dNTPs), a specific dideoxynucleotide triphosphate (ddNTP) is added to every reaction. The extension of a newly synthesized DNA strand terminates every time the corresponding ddNTP is incorporated. As the ddNTP is present in minute amounts, the termination happens rarely and stochastically, resulting in a cocktail of extension products where every position of an .N. base would result in a matching product terminated by incorporation of ddNTP at the 3´ end.

Deoxy versus dideoxy

DNA synthesis

Sanger method( manual); Reaction Components; There should be four reaction tubes A, C,G ,T ,each contains the following; The DNA to be sequenced ,as a single strand (the template). A primer complementary to the 3’ end of only one strand template DNA. All four normal (deoxy) nucleotides in ample quantities ,dATP , dGTP ,dCTP , dTTP . DNA polymerase ( Taq) . Each tube is then “spiked” with a different radioactively labeled ddNTP ,(ddATP for tube A, ddCTP for tube C, ddGTT for tube G, or ddTTP for tube T). concentration of ddNTP should be 1% of the concentration of dNTP. The logic behind this ratio is that after DNA polymerase is added, the polymerization will take place and will terminate whenever a ddNTP is incorporated into the growing strand. If the ddNTP is only 1% of the total concentration of dNTP, a whole series of labeled strands will result .

Composition of a DNA sequencing reaction (ddNTP) (The primer has a nucleotide sequence that is complementary to the 3 end of the region to be copied and is required so that DNA polymerase initiate DNA replication))

Sanger Method Procedure(manual); The targeted DNA fragment is amplified (multiplied) by PCR, the template DNA that is to be sequenced should be denatured producing single stranded DNA , this single stranded DNA (template) is then mixed with a primer complementary to the template DNA and the four normal dNTPs . This mixture is then split into four different tubes that are labeled A, C, G, and T. Each tube is then “spiked” with a different radioactively labeled ddNTP (ddATP for tube A, ddCTP for tube C, ddGTT for tube G, or ddTTP for tube T). DNA polymerase is added and using the DNA template and its’ complementary primer, the synthesis of new strands of DNA complementary to the template begins. Occasionally a dideoxynucleotide is added instead of the normal deoxynucleotide and synthesis of that strand is terminated at that point.

Sanger Method Procedure(manual); In the tube containing ddATP, some percentage of newly synthesized molecules will get a ddATP in each place that there is a T in the template DNA. The result is a set of new DNA molecules in tube A, each of which ends in an A . A similar type of reaction occurs in the three other tubes to result in molecules that end in C, G, and T in tubes C, G, and T respectively. After 20 - 30 cycles of the PCR heating and cooling, the resulting mixture will contain a series of fragments of different lengths depending on how many bases had been added to the chain before one of the dideoxynucleotides sneaked in and blocked further growth.

Sanger Method Procedure(manual; After the synthesis reactions are complete, the products of the four different tubes are denatured . Then loaded onto four adjacent lane of a polyacrylamide gel and the different fragments are separated by size . The sequencing gel is able to resolve fragments that differ in size from each other by only one base. All fragments in lane A will end in an A, fragments in lane C will all end in a C, fragments in lane G will all end in a G, and fragments in lane T will all end in a T. After electrophoresis ,the fragments are visualized by exposing the gel to photographic film (Remember that one nucleotide was radioactively labeled). The sequence of the DNA is read from the gel by starting at the bottom and reading upward.

Sanger Method Procedure(manual); For example if we looked at only the "G" tube, we might find a mixture of the following products:

Sanger Method Procedure(manual);

DNA Sequencing procedure The content of the reaction tubes are then transferred to four lanes of an electrophoresis gel The oligonucleotides are separated by size and nucleotide type The shortest oligonucleotide moves furthest down the gel.

DNA Sequencing procedure Reading from the bottom to top one base at a time, provide the correct DNA sequence

Automated DNA Sequencing

Automated Sequencing; With the many advancements in technology that has been achieved since Sanger´s manual method has been discovered and used, a new technology, Automated sequencing , has emerged to replace the manual method which is based on the same principles of Sanger's method. Automated sequencing has been developed so that more DNA can be sequenced in a shorter period of time.

Automated Sequencing; With the automated procedures all four dideoxy reactions are done in a single tube containing all four ddNTP's .This is possible because each ddNTPs is labeled with a different flourescent dye. Therefore the dye present in each synthesized fragment corresponds to the dye attached to the dideoxynucleotide that was added to terminate the synthesis of that particular fragment. The contents of the single tube reaction are loaded (after denaturation )onto a single lane of a gel and electrophoresis is done.

Automated Sequencing A flourimeter and computer are hooked up to the gel and they detect and record the dye attached to the fragments as they come off the gel. The sequence is determined by the order of the dyes coming off the gel.

The four reaction mixture are pooled Through gel electrophoresis, the mixture of the fragment can be ordered according to size, with the smallest fragments migrating the fastest in the gel

The last incorporated nucleotides in these fragments provide the complementary sequence to the template DNA strand. This complementary sequence is 5-GAACAGTTGCATCAG-3, and therefore the template strand consists of the sequence 5-CTGATGCAACTGTTC-3

An automated device scans the gel An automated device scans the gel. A laser beam strikes the band on the gel, causing the tags on the DNA to fluorescent colors and determines the corresponding sequence of the DNA

Automated Sequencing Automated DNA sequencers can read upto 96 DNA sequences in a 2 hours period, which is extremely fast as compared to manual DNA sequencing. Automated DNA sequencing has the following advantages over manual DNA sequencing: (1) radioactivity is not used, (which represents a health hazard to researchers). (2) gel processing after electrophoresis and autoradiography are not needed. (3) the tedius manual reading of gels is not required as data are processed in a computer, (4) the sequence data is directly fed into and stored in a computer, (5) the separation of the same reaction products can be repeated to recheck the results in cases of doubt since they can be stored for a long period of time, and (6) it is extremely fast.