Molecular Techniques and Methods

Dideoxy Chain-Termination Sequencing of PCR products

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

The amplification of target DNA by PCR followed by the direct sequencing of amplified DNA has emerged as a powerful strategy for rapid molecular genetic analysis. Using this strategy, time-consuming cloning steps can be completely bypassed and the sequence of the target DNA can be determined directly from a crude biological sample.

This protocol consists of three steps.
(1) The double-stranded, PCR-amplified DNA is denatured to single strands and the sequencing primer is annealed to the complementary sequence on one of the template strands.
(2) The annealed primer is extended by 20-80 nucleotides by DNA polymerase, incorporating multiple radioactive labels into the newly synthesized DNA.
This step is performed under nonoptimal reaction conditions so that the enzyme acts in a low-processive fashion, synthesizing only short stretches of DNA.
(3) The labeled DNA chains are extended and terminated by the incorporation of the ddNMP.




MATERIALS AND SOLUTIONS

10 x Denaturation Solution (1 ml)
2 M NaOH ---------------------------------- 400 ul of 5 M NaOH
2 mM Na2-EDTA --------------------------- 4 ul of 0.5 M Na2-EDTA
Distilled H2O -------------------------------- 596 ul


5 x Sequenase Buffer (1 ml)
200 mM Tris-HCl (pH 7.5) ------------------ 200 ul of 1 M Tris-HCl
100 mM MgCl2 ----------------------------- 100 ul of 1 M MgCl2
250 mM NaCl ------------------------------- 50 ul of 5 M NaCl
Distilled H2O -------------------------------- 650 ul


5 x Labeling Mix (1 ml)
7.5 uM dGTP ------------------------------- 7.5 ul of 1 mM dGTP
7.5 uM dCTP ------------------------------- 7.5 ul of 1 mM dCTP
7.5 uM dTTP ------------------------------- 7.5 ul of 1 mM dTTP
Distilled H2O -------------------------------- 977.5 ul


ddG Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddGTP -------------------------------- 8 ul of 1 mM ddGTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddA Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddATP -------------------------------- 8 ul of 1 mM ddATP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddT Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddTTP -------------------------------- 8 ul of 1 mM ddTTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


ddC Termination Mix (1 ml)
80 uM dGTP -------------------------------- 8 ul of 10 mM dGTP
80 uM dATP -------------------------------- 8 ul of 10 mM dATP
80 uM dCTP -------------------------------- 8 ul of 10 mM dCTP
80 uM dTTP -------------------------------- 8 ul of 10 mM dTTP
8 uM ddCTP -------------------------------- 8 ul of 1 mM ddCTP
50 mM NaCl -------------------------------- 10 ul of 5 M NaCl
Distilled H2O -------------------------------- 950 ul


Enzyme Dilution Buffer (1 ml)
10 mM Tris-HCl (pH 7.5) ------------------- 10 ul of 1 M Tris-HCl
5 mM DTT ---------------------------------- 5 ul of 1 M DTT
0.05% BSA --------------------------------- 50 ul of 1% BSA
Distilled H2O -------------------------------- 935 ul


Formamide Loading Buffer (1 ml)
100% Formamide --------------------------- 1ml
20 mM Na2-EDTA ------------------------- 40 ul of 0.5 M Na2-EDTA
0.05% BPB -------------------------------- 10 ul of 5% BPB
0.05% Xylene cyanol ----------------------- 10 ul of 5% Xylene cyanol




PROCEDURES

Preparation of PCR-amplified DNA for Cycle Sequencing Reaction

  • The purity of the template will affect the quality of the sequence data.

    1. The PCR-amplified DNA should be first purified by QIAquick column chromatography to remove unused dNTPs and primers.
  • It is possible to use 1-2 ul of the unpurified PCR product directly for sequencing. However, the overall quality of the sequence ladders generated is not as good as with the purified DNA. Therefore, the purification of amplified DNA is recommended.
  • It is desirable to have a template DNA concentration of at least 0.1 pmole for each set of sequencing reactions to generate high-intensity sequencing ladders.


    Sequencing Reaction

    2. Mix the following in a microfuge tube.

    PCR-amplified DNA
    1 pmole
    Sequencing Primer
    10 pmoles
    5 x Sequenase Buffer
    2 ul
    Add disitilled H2O to make a final volume of
    10 ul


    3. Incubate the samples in a heat block for 8 minutes at 94-96oC.

    4. Chill the tubes on ice for 1 minute.

    5. Centrifuge in a microfuge at 10,000 rpm for 10 seconds.

    6. Transfer the tubes to ice, and immediately proceed to the next step.

  • The above annealing conditions are applicable only to double-stranded, PCR-amplified DNA templates.
  • For single-stranded templates, such as the products of asymmetric PCR, the gradual cooling technique is preferred. In this procedure, the above mixture is incubated for 6 minutes in a 65oC heat block and is gradually cooled to 30oC by turning off the heat block.


    7. To the above mixture on ice, add the following.

    1 x Labeling Mix (diluted by H2O)
    2 ul
    [a-32P]dCTP (10 uCi/ ul, 3000 Ci/mmole)
    5 uCi
    0.1 M DTT
    1 ul
    8 x Diluted Sequenase (diluted by Enzyme Dilution Buffer)
    2 U
    Final volume
    15.5 ul


    8. Dispense 2.5 ul of each Termination Mix in the microwell titerplate.
  • Preincubated for 5 minutes at 37oC.

    9. Terminate raction by adding 3.5 ul Reaction Mix from step 6 in each well containing 2.5 ul Termination Mix.
  • Mix by pipetting up and down ONE time.

    10. Allow the extension and chain-termination reactions to proceed for 5 minutes at 37oC.

    11. Terminate the reaction by adding 4 ul of Formamide Loading Buffer.
  • If the samples are not used immediately, they can be stored frozen at -70oC for about a week. However, it is preferable to use the samples within 2 days.

    12. Heat the samples for 3 minutes in an 90oC heat block, and load a 2 ul aliquot of the sample in each lane of a Sequencing Gel.




    NOTES

  • For sequencing GC-rich templates, dGTP should be replaced with 7-deaza-dGTP to overcome compression artifacts that are known to occur during sequencing gel electrophoresis. dGTP can also be replaced with dITP, but dITP may not be a good substrate for all DNA polymerases.


  • Manipulation of the Size Range of Sequence Ladders
    Most of the compositions of dNTP/ddNTP mixtures reported in the literature are optimized to generate sequence ladders of high intensity and uniformity in the range of 50-200 nucleotides from the 3'-end of the primer. Generally, the sequence ladders closer to the primer (1-50 nucleotides from the 3'-end of the primer) are of low intensity under these conditions.
    If high-intensity sequence ladders are desired very close to the primer (in the 1-100 nucleotide range), Mn2+ can be added to the sequencing reactions. DNA polymerases incorporate ddNTPs about 5-10-fold more frequently in the presence of Mn2+. Because Mn2+ effects are seen in the presence of Mg2+, no changes to the basic protocols are required other than the addition of Mn2+. Different polymerases require different Mn2+ concentrations for optimal results. A Mn2+ concentration of 3.5 mM is optimal for Sequenase for increasing the frequency of terminations by about fivefold.
    If it is desired to generate sequence ladders far from the primer (in the 200-400-nucleotide range), the dNTP/ddNTP ratios should be increased by simply adding an appropriate aliquot (this varies depending on the polymerase used) of a dNTP stock solution to the extension-termination mixture. This decreases the frequency of terminations, and increases the average length of the chain-terminated products.


  • Carryover Nucleotides and Primers
    In a standard PCR, about 200 uM each of four dNTPs and 50 pmoles each of two primers are used to amplify several micrograms of target DNA. Of these, >97% of dNTPs and >90% of primers remain unused at the end of PCR. Unless these are removed, direct use of PCR-amplified DNA as a sequencing template results in the carryover of unused dNTPs and primers to the sequencing reactions. These interfere with the sequencing reactions in the following ways:
    (1) The carryover dNTPs alter the dNTP/ddNTP ratios that are required for optimal chain-termination reactions.
    (2) Since the carryover primers can also prime DNA synthesis, they compete with the sequencing primer and titrate out polymerase as well as dNTPs and ddNTPs.
    (3) As a result of priming by carryover primers, a mixture of sequence ladders is generated by the Sequenase protocol because the DNA synthesized from the PCR primers is also radioactively labeled. As a consequence of the above interferences, the sequence ladders generated are of low intensity with high background.
    It is difficult to decipher the DNA sequence from such ladders; therefore, it is essential to remove the unused dNTPs and primers from PCR mixtures. A number of strategies have been reported for the separation of low-molecular-weight dNTPs and primers from the high-molecular-weight, PCR-amplified DNA. These include differential precipitation, ion-exchange chromatography, gel filtration, and streptavidin chromatography. Of these, ion-exchange chromatography may be the best way to remove quantitatively the low-molecular- weight dNTPs and primers from the high-molecular-weight PCR-amplified DNA. QIAquick spin column separation gives clean DNA templates and generates high-intensity sequence ladders. These columns are designed to resolve either the single stranded or the double-stranded PCR-amplified DNA from dNTPs and PCR primers that are less than 50 nucleotides long.


  • Heterogeneity of DNA Template
    PCR-amplified DNA is a product of virtually billions of in vitro priming and extension reactions. Therefore, inherent in the PCR process is the amplification of heterogeneous DNA in addition to the unique target DNA. These heterogeneous DNA molecules differ in sequence and size, and arise as a result of secondary reactions. These include (1) partial products generated by the premature termination of DNA synthesis, (2) mosaic products generated by random intermolecular recombination between target DNA strands, and (3) multiple products generated as a result of priming at sequences that have either accidental homology or functional relatedness to the target DNA of interest.

    Most of the standard PCRs amplify predominantly the unique target DNA, whereas the heterogeneous DNA constitutes a minor fraction. This fraction usually appears as a smear upon agarose gel electrophoresis and ethidium bromide staining. However, it is not uncommon to see that a major portion of the amplified DNA is constituted by heterogeneous DNA. This happens particularly when the copy number of starting sample is low or when PCRs are performed under low-stringency conditions. The heterogeneous DNA, depending on the amount present in the PCR-amplified DNA, accordingly contributes to the back-ground in the final sequence ladders.


  • Errors in the DNA Sequence Generated from PCR-amplified DNA
    It is known that the Taq DNA polymerase, which is the most widely used enzyme for PCR, lacks proofreading 3'-5' exonuclease activity and incorporates errors at a high frequency. Estimates of error frequencies under PCR conditions range from 1 error in 4,000 nucleotides synthesized to 1 error in 400 nucleotides synthesized. Therefore, it is possible that almost every molecule in a 1-kb size PCR-amplified DNA could have an error. The question then is whether direct sequencing of the mutant PCR-amplified DNA results in the generation of an inaccurate DNA sequence. The answer to this question is clear when one considers a worst-case scenario in which a hypothetical target DNA is amplified starting from a single DNA molecule and assuming that an error is incorporated in the very first cycle. Consequently, after the first cycle, one of the four DNA strands has a mutation. Upon further amplification of these four strands for about 25 cycles, the mutant sequence constitutes about 25% of the final amplified product. When this DNA is used as a template for sequencing, the final autoradiogram shows the mutant band only at one-third the intensity of the correct nucleotide band. Because the DNA sequence is read by subtracting the background, the nucleotide sequence deduced from such a sequence ladder is that of the correct nucleotide.

    The above scenario is one that is encountered rarely because, in most PCR experiments, the copy number of starting DNA is on the order of 103-105 molecules. Any errors incorporated in the initial cycles are randomized, and therefore, any specific mutant sequence constitutes only a minuscule fraction of the total product. The DNA sequence generated is a consensus sequence from millions of template DNA molecules, thus these errors are not accounted for in the final sequence. Therefore, in practice, the direct sequencing of PCR- amplified DNA does not result in the incorporation of errors. The sequence generated is accurate, despite the low fidelity of Taq DNA polymerase.

    Indeed, it is desirable to generate the DNA sequence by directly sequencing the PCR-amplified DNA rather than from a clone of the amplified DNA, because during cloning, a single DNA molecule out of billions of amplified molecules is selected. It is highly probable that the cloned molecule will contain a mutation as a result of PCR. Therefore, the sequence generated from a cloned DNA should be confirmed by comparing it with the sequence generated directly from the PCR-amplified product to ascertain the accuracy of the cloned sequence.


  • Summary
    High-intensity, low-background sequence ladders can be consistently generated directly from PCR-amplified DNA by
    (1) performing PCR under stringent conditions
    (2) purifying the PCR-amplified DNA from unused dNTPs and primers by Qiagen chromatography
    (3) performing cycle sequencing under stringent conditions
    (4) using a high-specific-activity, 5'-labeled sequencing primer for amplifying the chain-terminated products.


    KIT INFORMATION



    REFERENCES

  • Bachmann, B, Luke, W, Hunsmann, G (1990) Improvement of PCR amplified DNA sequencing with the aid of detergents. Nucleic Acids Res. 18: 1309.

  • Barr, PJ, Thayer, RM, Laybourn, P, Najarian, RC, Sela, F, Tolan, D (1986) 7-Deaza-2'- deoxy-guanosine-5'-triphosphate: Enhanced resolution in M13 dideoxy sequencing. BioTechniques 4: 428- 432.

  • Innis, MA, Myambo, KB, Gelfand, DH, Brow, MAD (1988) DNA sequencing with Thermus aquaticus polymerase and direct sequencing of polymerase chain reaction-amplified DNA. PNAS 85: 0436-0440.

  • Kusukawa, N, Uemori, T, Asada, K, Kato, I (1990) Rapid and reliable protocol for direct sequencing of material amplified by the polymerase chain reaction. BioTechniques 9: 66-71.

  • Rao, VB (1994) Direct sequencing of polymerase chain reaction amplified DNA. Anal. Biochem 216: 1-14.

  • Please send your comment on this protocol to "editor@MolecularInfo.com".
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