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MSI by PCR Workflow

How do I get started?

In MSI by PCR, DNA is first isolated from a tumor biopsy or resection sample and typically paired with a nonmalignant sample source for comparison. Following DNA isolation, specific mononucleotide sequences within the samples are amplified using polymerase chain reaction performed on a thermocycler. Microsatellite markers can be amplified in multiple, parallel reactions or multiplexed in a single reaction. When multiplexed primers for individual markers are fluorescently labeled, many different fragments of similar sizes can be detected in the same reaction.

Following amplification, the amplified fragments are resolved by size on a capillary electrophoresis instrument. Data analysis is then performed using specialized software developed for fragment analysis.

You’ll need:

  • DNA purification system (automated or manual)
  • Thermocycler
  • DNA amplification reagents (or commercially available reagents)
    • Marker sequence-specific DNA primers, fluorescently labeled
    • Thermal stable DNA polymerase
    • dNTPs
    • Magnesium
    • Salts and buffers to stabilize the polymerase and support extension from the primer
  • Capillary Electrophoresis Instrument capable of multi-dye fluorescence detection and consumables
  • Fragment Analysis Software

 

msibypcr

MSI Analysis Workflow

In a routine research laboratory, the time from sample input to MSI status determination can range from overnight to two days. With a PCR workflow, it is possible to have data ready for analysis overnight.

circle 1

DNA Isolation

5 hours including incubation

msi workflow maxwell purification
circle 2

Amplification

~3 hours

msi workflow amplification
circle 3

Capillary Electrophoresis

45 minutes

msi workflow ab3500 instrument
circle 4

Data Analysis

<1 hour

msi workflow analysis