1/12 Dideoxy DNA Sequencing

Transcription

1 1/12 Dideoxy DNA Sequencing Dideoxy DNA sequencing utilizes two steps: PCR (polymerase chain reaction) amplification of DNA using dideoxy nucleoside triphosphates (Figures 1 and 2)and denaturing polyacrylamide gel electrophoresis of PCR products (Figure 3). PCR is a technique in which a DNA template is copied repeatedly yielding large amounts of DNA fragments that are several hundred nucleotides long (Figure 3). In this procedure, a mixture is made of primer, DNA polymerase, deoxynucleoside triphosphates (dntps) and template. This mixture is subjected to a series of thermal changes, each of which is responsible for a specific step in the replication of the template. The DNA is first denatured at a temperature of 95 C. Following denaturation, the primer, which contains homology to the template, is allowed to anneal at a lower temperature. The annealing temperature of the primer is dependent on the size and G/C content of the primer used (we will use 54 C). Then the temperature must be adjusted to 70 C for extension (replication) of the DNA template by DNA polymerase. The DNA polymerase used is called Taq polymerase. Taq polymerase is isolated from a thermophilic bacterium called Thermus aquaticus and is used because the polymerase is thermally stable at 95 C. This cycle of temperature changes is repeated about 30 times resulting in amplification of the DNA template. In order to read the DNA sequence, the elongation of the template must be terminated at each base. This is accomplished by introducing dideoxynucleoside triphosphates (ddntps). Dideoxynucleoside triphosphates lack a hydroxyl residue at the 3 position of deoxyribose (Figure 5). They are incorporated by DNA polymerase into the growing DNA chain by their 5 triphosphate groups (phosphodiester bond). Incorporation of a ddntp into the chain terminates replication since there is no longer a free 3 OH; therefore, no more dntps can be catalyzed into the growing chain by the DNA Polymerase (Figure 3). 1

2 Figure 1. Polymerase Chain Reaction (PCR) leads to the amplification of the target sequence (single template strand) in preparation for dideoxy sequencing. Note that only the strand that contains the T7 promoter is being replicated. 2

3 Figure 2. Comparison of the structures of the normal DNA precursor 2 -deoxynucleoside triphosphate (dntp) and the chain-terminator 2,3 -dideoxynucleoside triphosphate (ddntp) used in DNA sequencing reactions. Note the absence of the 3 OH on ddntp. Figure 3. PCR Sequencing DNA by 2,3 -dideoxynucleoside triphosphate chain-termination procedure. The four reactions are carried out in parallel, each of which contains one of the 2,3 -dideoxy chain terminators. The DNA products of the four reactions are separated on a denaturing polyacrylamide gel. 3

4 In the example presented in Figure 3, four different reactions are initiated in four different microcentrifuge tubes. Each reaction (tube) contains template (GCATGATCGG), an infraredlabeled DNA primer, and deoxynucleoside triphosphate. In addition to these components reaction #1 contains a low concentration of dideoxyguanosine triphosphate (ddgtp), reaction #2 contains a low concentration of ddatp, reaction #3 contains a low concentration of ddctp, and reaction #4 contains a low concentration of ddttp. Now Taq DNA polymerase is added to each tube and the DNA is allowed to replicate using PCR. In the first reaction there are two sites at which the ddgtp can be inserted (due to a C in the template strand) and replication is terminated due to the addition of the dideoxy nucleoside triphosphate (see Figure 3). Since the concentration of ddgtp is low, most of the time the polymerase will insert dgtp instead of ddgtp. This will result in the production of two products from our template: CG dd and CGTACTAG dd. There are no other C s in the template and hence no other places where termination can occur by the addition of ddgtp. Each reaction will terminate at specific nucleotides depending upon which ddntp is in the reaction mixture. This results in DNA fragments in each reaction tube of varying length specified by the template. The four reactions are run (in four different lanes) on a 5.5% denaturing polyacrylamide gel that separates the DNA fragments according to their length. Differences as little as one nucleotide are easily distinguished. The gel contains 7.0 M urea (denaturant) and runs in 0.8X TBE (tris, borate, EDTA) buffer. The gel is run at approximately 2,000 volts at a temperature of about 45 C. Each band on the gel in the four lanes signifies a specific termination point in the newly synthesized strand. With the four reactions side by side, the DNA sequence can be read directly off the gel by a computer programmed to read the infrared dye (Figure 3). Sequencing from Plasmid Vectors (Thermo Sequenase Labeled Primer Cycle Sequencing Protocol for USB Kits) 1. PCR MACHINE SET UP: M13 Primer Pair T7 Promoter/Terminator Pair pbad Primer Pair 1) 92 C for 2 minutes 1) 92 C for 2 minutes 1) 92 C for 2 minutes 2) 92 C for 30 seconds 2) 92 C for 30 seconds 2) 92 C for 30 seconds 3) 54 C for 30 seconds 3) 51 C for 30 seconds 3) 47 C for 30 seconds 4) 70 C for 1 minute 4) 70 C for 1 minute 4) 70 C for 1 minute Repeat steps 2-4 for total Repeat steps 2-4 for total Repeat steps 2-4 for total 30 cycles 30 cycles 30 cycles 10 C soak 10 C soak 10 C soak 4

5 2. Combine DNA template and primer in a 1.5 ml tube: Template DNA g Primer (M13 Forward or M13 Reverse conc=1 pmol/ l) 3.0 pmol (1.5 pmol each) Thermo Sequenase Reaction Buffer 2.0 µl 2.5 mm each dntp nucleotide mix (with 7-deaza dgtp**) 1.0 µl Thermo Sequenase DNA Polymerase (4 units/ l) 2.0 µl (8 units) dh 2 O to bring final volume to 17.0 µl TOTAL VOLUME 17.0 µl Mix well. Add water last and mix the components well by pipetting the reaction up and down several times with the same tip. Simply tapping the tube is not sufficient to mix. **One of the perennial problems of DNA sequencing occurs when analyzing DNA segments that are G-rich. Basically, DNA structure dictated by Watson-Crick base pairing is disrupted in G- rich segments because of their ability to create inter and intra strand hydrogen bonding. This aggregation causes enzymatic disruption so sequencing and PCR experiments become highly problematical. The problem arises from extra hydrogen bonding forming at the N7 position of G. Traditionally, 7-deaza-G has been used to overcome these problems with some success because it eliminates intra strand hydrogen bonding. 7-deaza dgtp dgtp 3. Label a set of four 0.2 or 0.5 ml thin-walled PCR tubes A, T, G and C for each template/primer combination. 4. Add 4.0 µl of the A Termination mix (purple-capped tube) to the A tube(s), and the T Termination mix to the T tube(s). Do the same for the G and T tubes. 5. Add 4.0 µl of the appropriate template/primer combination (from step 2) to each A, T, G, and C tube and mix well. 6. Carefully seal the tubes, place in thermal cycler and start the program. 7. Pour a sequencing gel. 5

6 8. At the completion of the cycling program, add 3 µl of Stop Solution to each tube. 9. Denature samples at 92 C for 3 minutes and place on ice prior to loading the samples in the sequencing gel. 10. Load gel. 6