Molecular Cloning Fourth Edition, A Laboratory Manual, by Michael R. Green and Joseph Sambrook

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The material on this page is part of Chapter 10, which is shown in full as a preview on this site.

Chapter 10: Nucleic Acid Platform Technologies

Rando Oliver, Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605

INTRODUCTION

T7 Linear Amplification of DNA (TLAD) for Nucleosomal and Other DNA < 500 bp

(Protocol summary only for purposes of this preview site)

Protocol 2 has been used extensively in genomic localization analysis and appears to work quite well for typical applications in which DNA is sheared by sonication to 500 bp. However, when DNA is sheared to a population whose modal size is <500 bp, bias in the PCR step skews representation of some genomic loci (Liu et al. 2003). In addition, a subset of applications requires amplifying DNA populations that are smaller than 500 bp; a notable example is ChIP on mononucleosomal DNA, which is 150 bp long (Liu et al. 2005). In these circumstances, T7 linear amplification of DNA (TLAD) is preferred because it more accurately maintains uniform representation of short DNA fragments during amplification than does the amplification method described in Protocol 2.


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 Protocol 3: T7 Linear Amplification of DNA (TLAD) for Nucleosomal and Other DNA < 500 bp

Protocol 2 has been used extensively in genomic localization analysis and appears to work quite well for typical applications in which DNA is sheared by sonication to 500 bp. However, when DNA is sheared to a population whose modal size is <500 bp, bias in the PCR step skews representation of some genomic loci (Liu et al. 2003). In addition, a subset of applications requires amplifying DNA populations that are smaller than 500 bp; a notable example is ChIP on mononucleosomal DNA, which is 150 bp long (Liu et al. 2005). In these circumstances, T7 linear amplification of DNA (TLAD) is preferred because it more accurately maintains uniform representation of short DNA fragments during amplification than does the amplification method described in Protocol 2.

Amplification of double-stranded DNA by TLAD begins with the addition of a 3 tail of poly-thymidine to DNA by TdT. Second, the Klenow fragment of E. coli DNA polymerase is used, along with a T7-poly(A) primer, to generate a complementary strand that carries a 5 T7 primer. Finally, extension of the original T-tailed DNA strand yields a template suitable for T7-based transcription, which generates amplified RNA (aRNA). This technique avoids the jackpotting issues observed with PCR, in which an early amplification event leads to disproportionate representation of a particular sequence, because PCR follows exponential kinetics, whereas transcription is a linear amplification method.

This protocol takes some time to complete. Table 1 provides estimates of the time required for each procedure.


MATERIALS

It is essential that you consult the appropriate Material Safety Data Sheets and your institution's Environmental Health and Safety Office for proper handling of equipment and hazardous materials used in this protocol.

Recipes for reagents specific to this protocol, marked <R>, are provided at the end of the protocol. See Appendix 1 for recipes for commonly used stock solutions, buffers, and reagents, marked <A>. Dilute stock solutions to the appropriate concentrations.

Reagents

  • -Mercaptoethanol
  • Calf intestinal phosphatase (CIP) (New England Biolabs, catalog nos. M0290S or M0290L)
  • CoCl2 (5 mM)
  • Dideoxynucleotide tailing solution (8) (92 mM dTTP, 8 mM ddCTP) (Life Technologies)
  • dNTP mixture (5 mM) <A>
  • EDTA (0.5 M, pH 8.0) <A>
  • Ethanol (95100)
  • Klenow fragment of DNA polymerase I (New England Biolabs)
  • Mineral oil
  • NEB buffer 2 (10) (New England Biolabs)
  • NEB buffer 3 (10) (New England Biolabs)
  • NTP mixture (75 mM)
  • Reaction buffer, provided with kit
  • RNase-free H2O
  • RNase inhibitor
  • T7-A18B primer
    • The primer sequence is 5-GCATTAGCGGCCGCGAAATTAATACGACTCACTATAGGGAG(A)18[B], where B refers to C, G, or T. The primer should be HPLC, PAGE, or equivalent purification grade.
  • T7 RNA polymerase
  • Template DNA (maximum 500 ng per 10 L)
  • Terminal transferase (TdT) (New England Biolabs, catalog nos. M0315S or M0315L)
  • Terminal transferase buffer (5) (Roche, catalog no. 11243276103)

Equipment

  • MinElute kit (QIAGEN, catalog no. 28204)
  • RNase/DNase-free tubes (1.5 mL)
  • RNase-free PCR tubes (0.2 mL)
  • RNeasy Mini Kit (QIAGEN)
  • Rotary evaporator (e.g., SpeedVac)
  • Thermal cycler
  • Vacuum manifold (optional; see Step 17)
  • Water bath or heat block set to 37C


METHOD
CIP Treatment of Samples with Terminal 3-Phosphate Groups

Treatment of DNA with calf intestinal phosphatase (CIP) is only necessary if the source DNA has been sheared or treated with MNase. Treatment of the DNA with CIP removes 3-phosphate groups, leaving free hydroxyl groups on the 3 ends, which are necessary for efficient tailing by TdT. Failure to perform this step will likely reduce the yield of amplification products by 50.

  • 1. Set up the following reaction:
    Incubate the reaction for 1 h at 37C.
    • Each reaction can be scaled up to 100 L per tube.
  • 2. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
    • When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and reduce the volume of the DNA by drying, if necessary.

Tailing Reaction with Terminal Transferase

  • 3. Set up the tailing reaction:
    • Do not use the NEB buffer 4 supplied with the NEB terminal transferase because DTT in the buffer will precipitate the CoCl2 and inhibit the reaction. Use the cacodylate buffer (1 M potassium cacodylate, 125 mM Tris-HCl, and 1.25 mg/mL BSA at pH 6.6), either supplied with the Roche enzyme or purchased separately. Take precautions in handling this arsenic-containing buffer and use waste-disposal practices appropriate for your institution.
    • Do not freeze and thaw the dNTP mixes more than three times. Additional freezethaw cycles will degrade the dNTPs and will reduce the efficiency of the reaction.
    • Aim for 1 pmol of template molecules. The tested range is 2.575 ng of DNA per 10 L of reaction volume. Scale up the reaction volume accordingly for greater starting amounts. For ChIP samples, use a sensitive UV-Vis spectrophotometer or a fluorometer to quantify the amount of sample precisely. If the amount of DNA is unknown, scale up to a 20-L volume to ensure that there is enough TdT enzyme present for an efficient tailing reaction. Note that if insufficient enzyme is used, the efficiency of subsequent steps in the protocol will be significantly affected and result in significantly reduced yields (as little as 510 of normal expected yields).
    • We strongly suggest that the NEB terminal transferase be used for this protocol; TdT enzyme from other sources may not perform optimally. If using the Roche recombinant TdT, double the volume of enzyme.
  • 4. Add 12 drops of mineral oil to the top of the mixture to prevent evaporation of reactants during incubation. Incubate the reaction for 20 min at 37C.
  • 5. Stop the reaction by adding 2 L (per 10 L of reaction volume) of EDTA (0.5 M, pH 8.0).
  • 6. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
    • If the starting volume is 10 L, then add 10 L of water to bring the volume to 20 L before adding the reaction to the spin column. When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and dry the volume down if necessary.

Second-Strand Synthesis with Klenow Fragment Polymerase

  • 7. Set up the second-strand synthesis reaction:
    • If production of template-independent product is a significant problem, scale down the reaction volume while keeping the reagent concentrations (except for the T-tailed DNA) constant. See the end of the protocol for an example.
    • Do not freeze and thaw the dNTP mixes more than three times. Additional freezethaw cycles will degrade the dNTPs and will reduce the efficiency of the reaction.
    • NEB (early 2004) switched the supplied buffer for Klenow enzyme from EcoPol buffer to NEB buffer 2. This buffer should provide at least comparable yields to the old buffer and may actually increase yields up to 14.
    • Do not use mineral oil. Trace amounts of mineral oil appear to interfere with cleanup and in vitro transcription.
  • 8. Use the following program in a thermal cycler:
    • i. 2 min at 94C.
    • ii. Ramp from 94C to 35C at 1C/sec, then hold for 2 min to anneal.
    • iii. Ramp from 35C to 25C at 0.5C/sec.
    • iv. Hold for 45 sec at 25C (or up to 6 min).
      • During this time, add 1 L (5 U) of Klenow DNA polymerase. If necessary, centrifuge the tube to remove condensation from the top and sides of the tube.
    • v. 37C, 90 min.
    • vi. (Optional) 4C to temporarily halt enzyme activity until user returns to take reaction tubes out of cycler.
  • 9. Stop the reaction by adding 2.5 L of EDTA (0.5 M, pH 8.0) (the final concentration will be 45 mM).
  • 10. Clean up the DNA with a MinElute column. Follow the protocol supplied by the manufacturer. Elute the DNA in 20 L.
    • When working with <100 ng of DNA, the 10-L elution volume in the manufacturer's protocol may yield less than the 80 claimed by QIAGEN. Increase the elution volume to 1520 L, and dry the volume down if necessary. An elution volume of 20 L at this step increased yields by 3040 for a 50-ng sample.

In Vitro Transcription (IVT)

  • 11. In vitro transcription (IVT) requires that the double-stranded DNA (dsDNA) be in an 8-L volume. Dry down the eluate from 20 to 8 L in a rotary evaporator at medium heat for 1012 min (the drying rate is 1 L/min).
  • 12. Set up the in vitro transcription reaction in 0.2-mL RNase-free PCR tubes as follows:
    • If the IVT kit is new, combine the NTPs in one tube, then realiquot into four tubes. In the first three freezethaw cycles, yields drop 1015 after each cycle. If the NTPs go through more than three freezethaw cycles, each subsequent freezethaw cycle may drop the yield by as much as 50.
    • The buffer should be at room temperature. Adding cold buffer and dsDNA may cause the DNA to precipitate. If there is a precipitate, warm the buffer to 37C until the precipitate dissolves.
  • 13. Incubate the reaction overnight at 37C in a thermal cycler with a heated lid or in an air incubator.
    • The incubation can range from 5 to 20 h; typical is overnight, roughly 16 h.

Amplified RNA (aRNA) Purification Using RNeasy Columns

  • 14. Prepare the buffer (433.5 L per IVT reaction):
  • 15. Aliquot the mix into 1.5-mL RNase/DNase-free tubes.
  • 16. Transfer the contents of the IVT mix (from Step 13) to the RNase/DNase-free tube, and vortex gently and briefly.
  • 17. Add 250 L of 95100 ethanol, and mix well by pipetting. (Do not centrifuge!) Purify the aRNA through an RNeasy column by either the centrifuge method or with a vacuum manifold. aRNA Purification Using a Centrifuge
    • i. Apply the entire sample to an RNeasy Mini spin column mounted on a collection tube. Centrifuge the column for 15 sec at 8000g. Discard the flowthrough.
    • ii. Transfer the RNeasy column to a new 2-mL collection tube. Add 500 L of Buffer RPE (which must have ethanol added before use) and centrifuge for 15 sec at 8000g. Discard the flowthrough, but reuse the collection tube.
    • iii. Add 500 L of Buffer RPE onto the RNeasy column and centrifuge for 2 min at maximum speed.
    • iv. Remove the flowthrough, and pipette another 500 L of Buffer RPE onto the column. Centrifuge for 2 min at maximum speed.
      • This additional wash, which is not in the QIAGEN protocol, is necessary because of guanidinium isothyocyanate contamination in the eluted RNA.
    aRNA Purification Using a Vacuum Manifold
    • i. Apply the sample (700 L) to an RNeasy Mini spin column attached to a vacuum manifold. Apply vacuum.
    • ii. Shut off the vacuum, and pipette 500 L of Buffer RPE onto the RNeasy column. Apply vacuum.
    • iii. Repeat Step ii. Transfer the columns to 2-mL collection tubes. Centrifuge for 1 min at full speed.
    • iv. Return the column to the vacuum manifold, and add 500 L of Buffer RPE. Apply vacuum.
    • v. Transfer the column back to a 2-mL tube. Centrifuge for 1 min at full speed to completely dry the column.
  • 18. Transfer the RNeasy column into a new 1.5-mL collection tube, and add 30 L of RNase-free water directly onto the membrane. Centrifuge for 1 min at 8000g to elute the RNA. Repeat if expected; the yield is >30 g.
  • 19. Check the RNA concentration and purity by measuring the A260 and A260/A280.
  • 20. Proceed to Protocol 5 or 6 to add fluorescent label to the RNA.


TROUBLESHOOTING

In addition to the items below, it may be valuable to consult the troubleshooting section in the manufacturer's manual that accompanies the IVT kit.

Problem (Step 19): Amplified RNA appears to have been damaged by RNase.

Solution: Perform an IVT control, using 250 ng of the pTRI-Xef-linearized plasmid provided with the Ambion IVT kit. If not using the kit, use an appropriate amount of a dsDNA template that contains the pT7 promoter. Be sure that the chosen template has been used successfully as a template for T7 RNA polymerase. Yields should typically range from 100 to 140 g, limited by the 100-g binding capacity of the QIAGEN RNeasy column. If the yield from the IVT control template is poor, contamination with RNases can be assessed by running a 2 nondenaturing agarose gel in Tris-acetateEDTA (TAE) and ethidium bromide. An RNase-contaminated IVT sample will yield a smear of low-molecular-weight material. If RNase contamination is the cause, ensure that aerosol-barrier, RNase-free pipette tips are used, and that working surfaces are treated with RNase-decontaminating agents (e.g., RNaseZap; Ambion catalog no. 9780). This is particularly important when working with ChIP samples.

Problem (Step 19): Yields of aRNA are poor, but RNase contamination is not the cause.

Solution: If there is no RNase contamination detected, it is likely there may be problems with the IVT reaction conditions. Consider the following.

  • The NTP mix has gone through too many freezethaw cycles. NTPs are very sensitive to freezethaw cycles, and each one decreases the yield. Use a fresh IVT kit, and aliquot the NTP mix before use.
  • There has been excessive evaporation of the reaction volume during the incubation. The described IVT conditions (Steps 1113) were designed to limit evaporation and vapor volume during the long incubation period. Using mineral oil is not recommended because it may interfere with either the IVT reaction, the aRNA cleanup, or both.


DISCUSSION

The output of this protocol is amplified RNA (aRNA) suitable for labeling for microarray studies as described in Protocols 5 and 6. It is important to determine the amount of aRNA produced and calculate the mass amplification obtained. Typical amplifications result in at least a 200-fold increased mass yield. For example, 20 g of aRNA is synthesized from an input of 75 ng of DNA. Use a 12 agarose gel to assess the composition and quality of the amplified RNA. Unless knowing the size distribution is crucial, it is usually not necessary to run a denaturing gel. Within the resolution limits of an agarose gel, the amplified product may migrate 2040 bp more slowly on the gel. This shift is to be expected because of the addition of poly(A) tails, the tight size distribution of the poly(A) tail, and the sequence added by the T7 promoter. The size distribution of the poly(A) tail becomes particularly evident in amplification products produced from a single-size template, such as PCR products or a restriction-digested plasmid.

Second-Strand Synthesis with Limiting Primer Amounts

Occasionally a low-molecular-weight band may also appear near the bottom of the gel, at 100 bp. It has been observed under certain amplification conditions, usually when the concentration of starting material is significantly less than that of the primer during second-strand synthesis. In these cases, a substantial amount of small-molecular-weight material may be generated, which likely represents amplification product produced from IVT-valid template synthesized through the formation of primer dimers during second-strand synthesis. These products represent nonproductive material for downstream analysis, and if substantial amounts of this material are observed, then it is necessary to limit the amount of primer.

Limiting the amount of primer is important when amplifying from very small amounts of starting material. Not only will it decrease the amount of primer-dimer product, it can also increase the yield of the desired amplification product. Table 2 describes the single reaction volumes to use for a suggested mass range of starting material.

REFERENCES
Liu CL, Schreiber SL, Bernstein BE. . Year: 2003. Development and validation of a T7 based linear amplification for genomic DNA. BMC Genomics4: 19. doi: 10.1186/1471-2164-4-19.
Liu CL, Kaplan T, Kim M, Buratowski S, Schreiber SL, Friedman N, Rando OJ. . Year: 2005. Single-nucleosome mapping of histone modifications in S. cerevisiae. PLoS Biol3: e328. doi: 10.1371/journal.pbio.0030328.

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