DNA Sequencing and Fragment Analysis

Archive for the ‘DNA Sequencing – Troubleshooting’ Category

Double Sequence: A Common Problem in Sanger Sequencing

Sanger sequencing methodology requires template DNA to be relatively free from contaminating salts. Template quality is often determined electrophotometrically by loading the template on agarose gel. Agarose gel electrophoresis often shows the faster moving supercoiled DNA and slower moving nicked DNA found in a quality preparation. DNA samples produced by PCR amplification can be similarly tested by agarose gel electrophoresis. A good PCR product is viewed as a single clean band representing the desired length.

Another test is to measure absorbance spectrophotometrically at ultraviolet wavelengths 260 nm and 280 nm. A 260 nm / 280 nm ratio value of 1.8 is an indicator that the DNA is good.

Unfortunately, the result off the capillary sequencer shows double set of peaks. Because one set of peaks is directly over top the other, software cannot determine the correct bases to call as in figure 1. A result showing a double set of peaks is not as uncommon as many might believe.

What are the primary causes of double sequence?

Double Sequence Caused by Template Contamination

One of the early steps in plasmid DNA preparation is to select a desired colony and then inoculate liquid medium. In an overnight culture, the inoculated media would go through rapid bacterial cell division as the cells enter the log growth phase. Because the initial cells came from a single colony, it is believed that every cell is the identical product cloned from a single cell. However, this is not always the case. Colonies do not always come from a single cell. A colony could also be the product of multiple cells that failed to separate when spread on a plate. If the multiple cells are not identical, the sequence result could produce the double sequence shown in figure 1.

The region where double sequence begins is usually a restriction sight where a separate piece of DNA is inserted into a vector. One purpose of bacterial cloning is to produce copies of the inserted DNA. The clean sequence shown in figure 1 is vector sequence before the restriction sight. Despite differences in the two templates, the vector sequence is generally the same. The template contamination becomes apparent at the point where the new DNA has been inserted into the vector.

Often, the reverse or complimentary strand does not produce a double sequence and this is often confusing. If the DNA insert were relatively long, it would require sequencing the whole complimentary strand before double sequence is observed. The reason is simply because double sequence is caused by addition or deletion of an unknown number of bases at one restriction sight only. Therefore, both templates, the desired and contaminant have identical sequence in the complimentary direction.

PCR fragments could also produce double sequence. It is the result of errors in PCR amplification where two products are produced. The PCR fragments could produce two very similar sized products that appear as a single band once tested by agarose gel electrophoresis. Resolution using agarose gel is limited and does not separate DNA samples of very similar size.

Double Priming Results in Dual Amplification

Sanger sequencing is similar to PCR amplification. A primer anneals at the beginning of the region to be sequenced and Taq polymerase adds bases (dNTPs) in extension to produce and identical strand. A primer that matches two regions on either the insert or the plasmid could cause two separate amplifications simultaneously. The result of double priming is shown in figure 2.

Unlike template contamination, two separate products generally do not appear clean in the beginning. It does not require the primer to match identically to both annealing sights as long as the bases of the primer are similar with a match on the 3 prime ends. What happens if one annealing site is downstream from the other annealing site on the same strand of DNA? The downstream primer blocks bases extending from the first primer and the double sequence eventually becomes a single set of peaks.

Single Nucleotide Polymorphisms Could Also Cause Double Sequence

PCR amplification is used to determine potential heterozygous bases as single nucleotide polymorphisms (SNPs). It is one method for detecting mutations that could cause certain genetic diseases. The SNP likely could be observed as a base change in one of two identical alleles amplified together and is generally easy to spot from automated Sanger sequencing. A single base position is represented by two peaks to positively identify a SNP.

However, SNPs could also be the result of a base insertion or deletion (indel) as in figure 3. From the figure it is possible to follow two identical strands of sequence that are different by one base. Both alleles match until the GGCC region. However, one allele is missing the T-base and has shifted upstream by one base. The large clean peaks observed in the double peak region are simply the same base represented by two different products.

Preventing Double Sequencing in Sequencing Results

There are some cases where double sequence cannot be resolved as with PCR fragments that contain indels. However, certain steps could be taken to reduce conditions described in figure 1 and figure 2.

Plasmid preparations that produce double sequence at the point of insert can be prevented using a process called single colony isolation. Once the plasmid cells have been spread on a plate and grown, the select colony is isolated and spread on a second plate. Although the process requires additional time, it is worth the effort in cases when double sequence often occurs.

The process for preventing double sequence in PCR fragments is more complicated because PCR conditions need better optimization. It could be something as simple as reducing the amount of primer or number of cycles. Or require small adjustments in PCR conditions including temperature. Guidelines for PCR optimization will be a future topic.

Samples that produce double sequence as the result of double priming can be resolved by extending bases to the custom primer that was selected for sequencing. A better choice is to select a different primer even when double priming is caused from one of the possible universal primers present on a particular vector.

Double sequence result patterns are generally similar for each condition discussed here. Anyone is invited to present results similar to that presented. Questions are also welcome.

Please go here if you would like to download a

reprint for this article in pdf format


Nicked Plasmid DNA Prevents Automated Sanger Sequencing

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.