Molecular Techniques and Methods

Cycle 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. The crude sample can be cultured cells, bacteria, or a viral preparation. Furthermore, the copy number of target DNA in the sample can be as low as one to a few molecules of genomic DNA among a vast excess of contaminating nontarget DNA.

Each sequencing cycle consists of three steps.
(1) The PCR-amplified DNA is denatured to single strands.
(2) The annealing of a 32P-labeled sequencing primer (or a biotinylated primer) to the complementary sequence on one of the strands.
(3) The annealed primer is extended and chain-terminated by a thermostable DNA polymerase. The resulting partially double-stranded chain-terminated product is then denatured in the next sequencing cycle, releasing the template strand for another round of priming reactions, while accumulating chain-terminated products in each cycle. These steps are repeated for 20-40 cycles to amplify the chain-terminated products in a linear fashion.




MATERIALS AND SOLUTIONS

10 X Taq Sequencing Buffer (1 ml)
300 mM Tris-HCl (pH9.0) ---------------------- 300 ul of 1 M Tris-HCl
50 mM MgCl2 ---------------------------------- 50 ul of 1 M MgCl2
300 mM KCl ----------------------------------- 300 ul of 1 M KCl
0.01% Gelatin ----------------------------------- 10 ul of 1% Gelatin
Distilled H2O ------------------------------------ 340 ul


Termination Mix-A (1 ml)
2mM ddATP ------------------------------------- 20 ul of 100 mM ddATP
100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
Distilled H2O ------------------------------------ 976 ul


Termination Mix-C (1 ml)
1 mM ddCTP ------------------------------------ 10 ul of 100 mM ddCTP
100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
Distilled H2O ------------------------------------ 986 ul


Termination Mix-G (1 ml)
0.2mM ddGTP ----------------------------------- 2 ul of 100 mM ddGTP
100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
Distilled H2O ------------------------------------ 996 ul


Termination Mix-T (1 ml)
2mM ddTTP ------------------------------------- 20 ul of 100 mM ddTTP
100 uM dATP ----------------------------------- 1 ul of 100 mM dATP
100 uM dCTP ----------------------------------- 1 ul of 100 mM dCTP
100 uM dGTP ----------------------------------- 1 ul of 100 mM dGTP
100 uM dTTP ----------------------------------- 1 ul of 100 mM dTTP
Distilled H2O ------------------------------------ 976 ul


Stop Solution (1 ml)
10 mM NaOH ------------------------------------ 2 ul of 5 M NaOH
95% Formamide ---------------------------------- 950 ul of 100% Formamide
20 mM EDTA ------------------------------------ 40 ul of 0.5 M EDTA
0.05% Bromo Phenol Blue ------------------------ 10 ul of 5% Bromo Phenol Blue
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, particularly when cycle sequencing protocols are employed.

    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 cycle 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.


    Radioactive Labeling of Sequencing Primers

    2. Prepare a Master Labeling Mix containing the following components.

    Distilled H2O
    2.5 ul
    10 x T4 Polynucelotide Kinase Buffer
    2 ul
    [r-33P]ATP or [r-32P]ATP
    (10 uCi/ ul; 1,000-5,000 uCi/ mmole)
    2 ul
    T4 Polynucleotide Kinase
    10-20 U/ 2 ul
    Final Volume
    20 ul


    3. Label 2-5 pmoles of sequencing primer for each sequencing reaction as follow.

    Master Labeling Mix from step 2
    4 ul
    Sequencing Primer (2-5 pmoles/ 10-25 ng of a 17-mer)
    1 ul


    4. Incubate for 15 minutes at 37oC.

    5. Heat-kill the T4 Polynucleotide Kinase for 10 minutes at 80oC.

    6. The removal of unincorporated [r-32P]ATP, although not critical, is recommended.
  • This can be accomplished by gel filtration through a Biospin-10 column (Bio-Rad Laboratories).
  • The labeled primer can be stored at -70oC for at least 2 weeks.


    Cycle Sequencing Reaction and Gel Electrophoresis

    7. Prepare the Preraction Mix as follow.

    Labeled Sequencing Primer
    5 ul
    10 x Taq Sequencing Buffer
    4.5 ul
    0.1-0.2 pmole of purified PCR-amplified DNA
    70-140 ng of a 1-kb DNA
    Taq DNA Polymerase
    (No 3'-5' exonuclease activity)
    1-2.5 U/ 0.5 ul
    Add distilled H2O to make a final volume of
    36 ul


    8. Place tube in wet ice.

    9. Prepare one set of four sequencing reaction tubes.
    Add 2 ul each of Termination Mix.

    10. Pipette 8 ul of the Preraction Mix from step 7.

    11. Overlay 20 ul of mineral oil.

    12. Briefly centrifuge the tubes.

    13. Place the tubes in wet ice.

    14. Do PCR reaction as follow.

    1 cycle
    95oC
    1 min
    20 cycles
    95oC
    30 sec
    55oC (or, Tm-5oC)
    30 sec
    70oC
    60 sec
    10 cycles
    95oC
    30 sec
    70oC
    60 sec

  • Tm (oC) = [2 x (A+T)] + [4 x (G+C)]


    15. Stop reaction by adding 3 ul of Stop Solution.

    16. Heat at 90oC for 5 min.

    17. Load 2 ul each on a sequencing gel (6-8% denaturing-PAGE).

  • In 6% PAGE, bromo phenol blue moves like 26 nucleotides and xylene cyanol moves like 106 nucleotides.
  • In 8% PAGE, bromo phenol blue moves like 20 nucleotides and xylene cyanol moves like 80 nucleotides.




    NOTES

    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 (3-5 mM) for optimal results.

  • 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.
    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 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.

  • 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.

  • Other thermostable DNA polymerases such as the Vent polymerase and the Pfu polymerase exhibit proofreading exonuclease activity and reportedly incorporate errors 15- to 50-fold less frequently than the Taq DNA polymerase. Therefore, these polymerases should generate a better-quality PCR products particularly if the cloned DNA is also used for the expression and functional characterization of gene products.


    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, and
    (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".

  • Home
    MT&M
    Online Journal
    Hot Articles
    Order Products
    Classified