DNA Sequencing
The purpose of sequencing is to determine the order of the nucleotides of a gene. For
sequencing, we don't start from gDNA (like in PCR) but mostly from PCR fragments or
cloned genes.
Method for sequencing DNA
The need may arise for you to determine the sequence of the DNA you have amplified in
your PCR.
The chain termination method is often used.
In this method, a DNA strand is synthesized in vitro by the polymerase-catalyzed
incorporation of 2’-deoxynucleoside-5triphosphate (dNTPs) in the presence of a template
and annealed primer. Primer extension continues until the polymerase encounters and
incorporates a dideoxynucleotide (ddNTP) which cannot support continued DNA
elongation, and consequently, the chain is terminated.
By using four separate reactions each with a different ddNTP, (ddA, ddC, ddG, ddT) a
complete sequence can be obtained.
In this case the ddNTPs are labeled with fluorescent dye (Cy5.5 or Cy5) to ensure that
the properly terminated sequence is labeled and can be visualized by the laser reader on
the machine.
Enzymatic Pre-treatment of PCR product
Prior to sequencing your PCR product you need to purify the product from excess primer
and dNTP molecules.
This procedure involves treating PCR products with a combination of :Exonuclease (EX 1) removes residual single-stranded primers and any extraneous singlestranded DNA produced by PCR.
Shrimp alkaline phosphatase(SAP) removees any remaining dNTPs from thePCR
mixture which interfere with the labeling step of the sequencing process.
Steps:1-mix the PCR amplification mixture and enzymes as follows:
PCR product
16ul
Exonuclease 1, EX1 (10.0 U/ul)
0.3ul
Shrimp alkaline phosphates, SAP (2.0 U/u that l)
0.6ul
Dstilled water
3.1ul
Total volume
20ul
2- Incubate the mixture for 15 min at 37 c. It is convenient to do this in the PCR
machine.
3- Inactivate the exonuclease and phosphates by heating for 15 min at 80 c.
Your PCR product is now ready for the next step of preparation.
Sequencing reaction using CY5.5 dye –labelled ddNTP terminators.
1- Label four tubes A, C, G and T for the respective termination reaction.
2-Dispense 1ul of the corresponding CY5.5ddNTP termination mix into the appropriately
labeled tube .Cap the tubes to prevent evaporation.
3- For each template to be sequenced, prepare the following master mix in a micro
centrifuge tube:-
-DNA template (50-250fmol minimum)
up to 24 .oul
-Reaction buffer (concentrate)
3.5ul
-Primer 1uM (1pmo1\ul, 10ng\ul)
2.0ul
-Themo sequenase DNA polymerase (10U\ul)
2.0ul
-Dstilled water
x--ul
-Total volume
31.5ul
Note: The volume of DNA and primer added will depend on their concentration. Adjust
the amount of distilled water added so that the total volume of DNA, primer and water is
26.0ul. The total volume of the reaction mix should be 31.5ul.
4- Mix the contents of the master mix thoroughly by gently pumping the pipette and
centrifuge briefly. Aliquot 7ul of the mix into each the tubes labeled (A, C, G and T).
5- Mix each sequencing reaction thoroughly and centrifuge the tubes if necessary.
Place the tubes in the thermal cycler and run the appropriate program.
6- The sequencing program standard conditions are as follows:95 C
for 30s
60 C
for
30s
72 C
for
60-120s
(Usually 30 cycles)
7- After completion of the PCR program, centrifuge the tubes briefly to collect any
condensation that may occurred and place on ice.
Note: When sequencing single stranded DNA, the primer may anneal to the template with
reduced specificity while the tubes are on ice, and extension of the primer can occur as
the thermal cycler heats up during the first cycle. To minimize non specific extension
product, the cycle can be heated to 85-95°C (hot start). Alternatively, the cycler can be
pre-cooled to 4°C.
Cycle sequencing
In cycle sequencing, a repeated series of thermal denaturation, primer annealing, and
polymerization steps are used to linearly increase the amounts of product in DNA
sequencing reaction. Unlike PCR, wich uses two primers to increasetheamount of product
exponentially with the number of cycles, cycle sequencing uses asingle primer. If the
concentration of both dNTPs andddNTPs in the sequencing mixes are optimized, the
majority of products from a reaction will terminate within the resolving capacity of the
sequencing gel. When these product are separated in asuitable gel matrix using an
automated sequencing machine, sequencing data can be extracted from the fluorescent
emissions as the reaction products past the detection area.
Removal of unincorporated dye terminators using ethanol precipitation
1-
add 2ul of 7.5M ammonium acetate and 30ul of chilled 100% ethanol to each
tube. Mix thoroughly by vortexing and placing the tube on ice for 20min to precipitate
the DNA.
2-
Centrifuge the tubes in a microcentrifuge (approx 12000 rpm) for 20-30 min at
room temperature.
3-
Carefully remove each supernatant and wash the pellets with 200ul of chilled 70%
ethanol.
4-
Centrifuge for 5min. remove the supernatantand dry the pellet at room
temperature, taking care not to over dry it asit will be difficult to resuspend.
5-
Resuspend each pellet in 6ul of formamide loading dye and vortex vigorously 10-
20seconds to ensure complete suspension. Briefly centrifuge to collect the samples at the
bottom of the tubes.
6-
Just prior to loading the samples onto the gel, heat the samples to 70ºC for 2-3min
to denature, then place the sample on ice.