DNA Sequencing and Fragment Analysis

Development of simple-to-use purification kits from a number of commercial providers has simplified preparations of DNA samples used for automated DNA sequencing. The isolated DNA is generally clean with good yield. The template can then be quantified by spectrophotometry in preparations to submit to a sequence service provider. In addition, a spectrophotometric scan (220 nm to 310 nm) will indicate whether the plasmid DNA contains salts that could interfere with a sequencing reaction. Even though the template appears clean with good concentration, some samples fail. What could cause this? One possibility is that the DNA could be nicked.

What is Nicked DNA?

Plasmid DNA is characteristically a double-stranded supercoiled molecule. A restriction enzyme is often used to cut both strands linearizing the DNA molecule. A nick is an isolated break in one of the two strands keeping the supercoiled form intact (figure 1).

How Does DNA Become Nicked?

DNA can be enzymatically nicked for certain applications. However, nicked DNA is undesirable for automated sequencing. It is likely the DNA was damaged physically by shearing during purification. Causes of damage include excessive vortexing or pipetting that physically break the DNA. Over-drying can also damage supercoiled DNA. Most commercial kits warn against over-drying a DNA preparation.

How to Detect Nicked DNA

The best method for determining whether the DNA has become nicked is using agarose gel electrophoresis. Nicked DNA cannot be identified using spectrophotometry. A typical plasmid preparation is shown in figure 2. Plasmid preparations almost always have some nicked DNA. However, supercoiled DNA should have the darker band in the resulting gel. Lane 3 provides the best quality DNA for automated DNA sequencing. Samples loaded in lane 1 and lane 2 would most likely fail.

Why Does Nicked DNA Fail to Sequence?

The enzyme lock-key model for enzymes provides a simple explanation why nicked DNA does not sequence. The enzyme, Taq polymerase, needs to sit down on the DNA to catalyze the addition of the bases during extension. A nick in the DNA loosens the strands of the supercoiled DNA strands and the enzyme no longer fits the DNA molecule.

How to Prevent DNA from Becoming Nicked

As previously stated, plasmid preparation kits generally isolate both nicked and supercoiled DNA. It is possible to reduce the amount of damage during the purification procedure. One recommendation is to thoroughly read the directions provided for the kit. There are specific steps where shaking DNA in solution should be minimal. Often it could also be recommended to mix reagents and DNA gently. Excessive vortexing and pipetting should be avoided. And the final DNA isolate should not be overly dried.

DNA is relatively stable and could be useful for years when stored under the proper conditions. But DNA has some fragile characteristics as well. Special care in preparation could reduce damage that inhibits successful automated sequencing applications.


Comments on: "Nicked Plasmid DNA Prevents Automated Sanger Sequencing" (2)

  1. […] A good scanning profile does not always prove the DNA is good. Nicked DNA cannot be identified by scanning and may inhibit quality Sanger sequencing. (https://agctsequencing.wordpress.com/2012/02/16/nicked-plasmid-dna-prevents-automated-sanger-sequenci…) […]

  2. […] spectrophotometry does not reveal whether DNA is supercoiled or nicked as described previously (Nicked Plasmid DNA Prevents Automated Sanger Sequencing). Nicked DNA has been damaged mechanically. Aggressive vortexing during DNA purification is one […]

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