Unit III -rDNA -DNA sequencing methods.pptx

Rdna

1. DNA SEQUENCING

2. DNA sequencing & its Application 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 identified 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.

3. DNA sequencing Two main methods 1) Sanger dideoxynucelotide chain termination method (Commonly used manual methods) Manual methods Automated methods 2 ) Maxam & Gilbert Method - Chemical cleavage method (Not used nowadays)

4. Sanger’s Method Sanger sequencing, also known as the “chain termination method,” was developed by the English biochemist Frederick Sanger and his colleagues in 1977. This method is designed for determining the sequence of nucleotide bases in a piece of DNA (commonly less than 1,000 bp in length). Sanger sequencing with 99.99% base accuracy is considered the “gold standard ” for validating DNA sequences, including those already sequenced through next-generation sequencing (NGS). Sanger sequencing was used in the Human Genome Project to determine the sequences of relatively small fragments of human DNA (900 bp or less). These fragments were used to assemble larger DNA fragments and, eventually, entire chromosomes.

5. Principle of the Sanger Sequencing Method 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.

6. Principle of the Sanger Sequencing Method ddNTPs lacks hydroxyl group (-OH) at c3 of ribose sugar, so it cannot make phosphodiester bond with nest nucleotide, thus terminates the nucleotide chain Respective ddNTPs of dNTPs terminates chain at their respective site. For example ddATP terminates at A site. Similarly ddCTP , ddGTP and ddTTP terminates at C, G and T site respectively .

7. Reaction Components of the Sanger Sequencing Method There should be four reaction tubes A, C , G, T, each contains the following ; The DNA t o 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

8. Reaction Components of the Sanger Sequencing Method 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). C oncentration 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 . ( 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)

9. Components

10. Sanger Method Procedure The Sanger sequencing method consists of 6 steps : (1) The double-stranded DNA ( dsDNA ) is denatured into two single-stranded DNA ( ssDNA ). (2) A primer that corresponds to one end of the sequence is attached. (3) Four polymerase solutions with four types of dNTPs but only one type of ddNTP are added . ( 4) The DNA synthesis reaction initiates and the chain extends until a termination nucleotide is randomly incorporated . (5) The resulting DNA fragments are denatured into ssDNA . (6) The denatured fragments are separated by gel electrophoresis and the sequence is determined .

11. Sanger Method Procedure 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 . Sometimes, a dideoxynucleotide is added instead of the normal deoxynucleotide and synthesis of that strand is terminated at that point.

12. Sanger Method Procedure 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.

13. Sanger Method Procedure 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.

14. 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.

15. Sanger’s Method Reading from the bottom to top one base at a time, provide the correct DNA sequence

16. Automated DNA 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 .

17. Automated DNA 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.

18. Automated DNA 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 .

19. Automated DNA Sequencing 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

20. Automated DNA Sequencing The last incorporated nucleotides in these fragments provide the complementary sequence to the template DNA strand.

21. Automated DNA Sequencing 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

22. Automated DNA Sequencing

23. Automated DNA Sequencing- Advantages 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 tedious 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.

24. Maxam - Gilbert Sequencing Method It is a method by which the sequence of a DNA fragment is identified by using the chemicals, that cut the DNA at specific points This method is based on nucleobase - specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides Also called chemical degradation method of DNA sequencing Develop by Allan Maxam and Walter Gilbert in 1976 to 1977

25. Maxam - Gilbert Sequencing Method - Procedure Lets suppose it is a fragment of double stranded DNA & we don't know its sequence STEP : 1 As the sequence of both strands are unknown , but if we find out the sequence of one strand we would get to know sequence of other one as well. High pH or high temperature At first the double stranded DNA is separated into two single strands by applying high pH or high temperature.

26. Maxam - Gilbert Sequencing Method - Procedure STEP : 2 Run the single stranded fragment on gel Lighter fragment will move faster than the heavy fragment band The band having larger number of purines ( A, G) would be heavier Step:3 Take one fragment band from the gel I ncorporate Radioactive Phosphate 32-Po4 enzymatically at the phosphate at 5 prime end

27. Maxam - Gilbert Sequencing Method - Procedure Step:4 Radioactive labelling Now put all the radioactive labelled fragments in four tubes . Step:5 Chemical degradation Tube 1: increase temp and pH ( by adding NAOH) that would cause fragments to break down. Dimethyl sulfate will be added that would make cuts at Adenine and Guanine positions. Tube 2: dimethyl sulfate and dilute HCl will added that would cuts the fragment at Adenine position Tube:3 Reagents hydrazine and piperidine are added that would cuts the fragment at position Cytocine and thyamine Tube:4 In the last tube, Hydrazine, Piperdine and NACL is added that would cuts the fragments Cytosine position. After chemical degradation we will get the following radioactively labelled fragments from each tube

28. Maxam - Gilbert Sequencing Method - Procedure Step : 6 GEL ELECTROPHOROSIS All fragments from each four tubes are pour in gel Four wells will be make on gel In first well, fragments from 1st tube is poured & in 2nd well fragments from 2nd tubes and so on. Fragments will separate on gel according to size. Smaller fragments would move farther than larger fragments. After placing radioactive film on top of gel, radioactive labelled fragments would emit a spot at their position.

29. Maxam - Gilbert Sequencing Method - Advantages Directly read purified DNA No premature termination due to DNA sequencing , so no problem with polymerase to synthesize DNA. Used sequence heterogeneous DNA as well as homopolymeric sequences Used to analyse DNA protein interaction i.e footprinting Used to analyse epigenic modification and nucleic acid structure to DNA.

30. Maxam - Gilbert Sequencing Method - Disadvantages Not widely used. Use of toxic chemicals and extensive use of radioactive isotopes, highly poisonous and unstable Cannot read more than 500bp. Setup is quite complex, technically complexity It is difficult to make Maxam Gilbert based DNA sequencing kit. Read size decrease with incomplete cleavage reactions

31. Maxam - Gilbert Sequencing Method - Limitations Gel electrophoresis is limited to700 to 900 bp , with 400 to 500 more commonly attained The first 15 t0 40 bp are often difficult to interpret Time acquire process i.e time spend on loading and running samples on gels, autoradiography and A nalysis, all lower the amount of DNA that can be sequenced

32. Maxam - Gilbert Sequencing Method It is a method by which the sequence of a DNA fragment is identified by using the chemicals, that cut the DNA at specific points Also called chemical degradation method of DNA sequencing Develop by Allan Maxam and Walter Gilbert in 1976 to 1977