DNA Sequencing and Fragment Analysis

Posts tagged ‘Agarose Gel Electrophoresis’

Automated Fragment Analysis Improves Accuracy Over Manual Methods

Automated capillary sequencers are also capable of performing fragment analysis applications. The process is similar to sequencing because fluorescent dyes are used to label products amplified by PCR. Fragment analysis is often performed manually loading amplified products on agarose gel. It is a relatively simple and cost effective method for conducting fragment analysis. However, there are also limitations. First, there is no method for accurately calculating fragment sizes even with a size ladder. Second, multiplexing is not possible when product sizes from multiple primer pairs (markers) are similar in size. Finally, agarose gels does not have the resolution capabilities that capillary electrophoresis has. Capillary electrophoresis, used in forensics fingerprinting, provides remarkable accuracy to fragment analysis applications.

Automated Fragment Analysis Uses Color Fluorescent Dyes

The Polymerase Chain Reaction (PCR) first amplifies samples that will be compared by fragment analysis. For manual applications, the forward and reverse primer are simply unlabeled oligonucleotides. For automated capillary equipment the forward primer contains a fluorescent label on the 5’end. The amplified product will also be labeled following PCR because the product includes both primers (figure 1). The forward and reverse primers (markers) determine the region and base pair size of the resulting amplification.

There are advantages to using fluorescent labels in the amplification process. Capillary analyzers recognize fluorescent emission wavelengths (different colors). This adds the capacity to multiplex more than one set of primers in an amplification reaction. For example, forensic science currently runs 16 separate markers in one multiplexed reaction. Markers are labeled with four different fluorescent dyes. Fragment sizes add a second parameter because fragments with the same dye, but amplified with different markers, never overlap.

A Standard is Added to Every Sample

Another parameter that increases accuracy and resolution in automated fragment analysis applications is the addition of a given standard to every sample. The base pair sizes of test samples are calculated using this standard. Motility variance that could occur from one capillary to the next is eliminated because a standard curve is determined for each capillary. Each fragment in the standard would need to be labeled with fluorescent dye. However, the standard uses a unique label not used for any of the samples, typically ROX or TAMRA.

Resolution of Automated Capillary Fragment Analysis is 1 Base Pair

The resolution capability of capillary fragment analysis is 1 base pair. The same technology that separates each base in sequencing is also applied to fragment analysis. A single base pair separation is nearly impossible using an agarose gel (figure 2). Fragment size accuracy is also limited by manual methods because there is no way to view the standard and sample fragments loaded in the same lane.

The introduction of slab gel sequencers improved capacity to perform fragment analysis over manual agarose methods. Development of capillary analyzers has brought greater sensitivity and speed to this well-established process. Very little genomic DNA is now required to perform automated fragment analysis. Although science has developed new methods for sequencing, fragment analysis still remains an efficient and cost effective method to analyze genomic variability; particularly related to genetic disease and forensics.

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DNA Fragments Resolve Better on Correct Percent Agarose Gel

An interesting article was posted March 25, 2011 on BitesizeBio.com titled 5 Ways to Destroy Your Agarose Gel. Every researcher may have made some of these common mistakes at one time. The five ways provided are…

  1. Use water instead of buffer for the gel or running buffer.
  2. Forget to add ethidium bromide
  3. Use the wrong percentage (or type) of agarose.
  4. Switch the leads from the power source.
  5. Drop the gel on the way to the imager.

The focus of this article is to explain the importance of using the correct percentage gel. In many genetic analysis applications a 1% agarose gel is commonly used to test plasmid preparations and PCR fragments. However, the resolution of the 1% gel may not sufficiently resolve smaller DNA products.

Percent Agarose Determines Pore Size

Agarose gel electrophoresis is a form of chromatography. The gel provides the stationary phase and electrical current provides the mobile phase. Charged particles such as DNA will migrate towards the positively charged anode in response to an electrical current across the gel. The gel provides the resistance against DNA migration. Smaller fragments move more rapidly than larger fragments.

Resistance is directly proportional to the porous nature of an agarose gel. Smaller pores provide more resistance. Increasing the percent of the gel decreases the size of the pores. When the pore sizes are too large small DNA fragments migrate together and do not become separated (figure 1). This figure illustrates why large DNA fragments should not be run on an agarose gel with small fragments of DNA.

Correct Percent of Agarose Depends on the Size Products Tested

The correct percent agarose gel is dependant on the size of the fragment that will be tested. Plasmid DNA preparations that are 5 kb to7 kb resolve well on a 1% gel. Large PCR fragments that are similar in size to plasmid DNA could also resolve on a 1 % percent gel. However, small PCR fragments that require smaller pore size for better resolution require a higher percent gel. General guidelines for mixing the correct percent gel are provided in table 1.

For small PCR fragments less than 500 bases in size, it is best to use a two percent gel. This will increase the run time. However, it will also improve resolution of fragments that are similar in size and may not resolve on lower percentage gels.