Overview

Real-time reverse transcription (RT) polymerase chain reaction (PCR) is the most sensitive and reliable method for the detection and quantitation of nucleic acids levels. The Biotechnology Lab has acquired the ABI 7900 sequence detection system which allows real time detection of PCR products as they accumulate during PCR and thus enables the accurate quantitation of DNA and RNA over a wide dynamic range. The ABI 7900 instrument can be used for various applications such as quantitation of gene expression, allelic discrimination (SNP detection) and pathogen detection.

Quantitative Real-Time PCR

Allelic Discrimination Assay (SNP Genotyping)

Primer and Probe Design Guidelines

Primer Express designs probes and primers suitable for the universal thermal cycling conditions specified by ABI.

Notes

  • Blast the sequence of the entire gene and identify a region which is specific for that gene. Use this sequence for designing primers and probes using primer express.
  • Following guidelines are used automatically (by default) by Primer Express.
  • Select the Probe first ant then design primers.

Target Sequence and Amplicon Size Guidelines

  • Primers and probes should be selected in a region with G/C contents of 20-80%. Lower G/C content is preferred.
  • Design primers to amplify short segments of DNA within the target sequence. The recommended amplicon size is 50 to 150 bp.
  • Design at least one primer and/or probe which crosses one exon junction. The primers thus created would amplify mRNA (or cDNA made from it), but not genomic DNA. Use of Junction Annotation tool from Primer express which allows the user to mark exon junctions and selects at least one primer crossing at least on exon junction.

TaqMan Probe Design Guidelines

  • The melting temperature (Tm) of the probe should be 68-70 0 C.
  • Keep G-C contents in the 30-80% range.
  • Avoid runs of an identical nucleotide. This is true especially for G, where runs of four or more Gs should be avoided.
  • Do not put Gs on the 5. end.
  • Select the strand that gives the probe more Cs than Gs. Primer express does not automatically screen for this feature. It needs to be done manually.
  • If the TaqMan probe is designed for allelic discrimination, the position of the polymorphic site (mismatch) should be approximately in the middle third of the sequence.

Primer Design Guidelines

  • Design primers as close to probe as possible
  • Keep G-C contents in the 30-80% range.
  • Avoid runs of an identical nucleotide. This is true especially for G, where runs of four or more Gs should be avoided.
  • The melting temperature (Tm) of the primer should be 58-60 0 C.
  • The total number of Gs and Cs in the last five nucleotides at the 3. end of the primer should not exceed two. The software does this automatically if the option is selected.
  • Primers should scan exon-exon junction. Contaminating genomic DNA will not be amplified by these primers.
  • Use primers that contain dA nucleotides near the 3. ends so that any primer-dimer generated is efficiently degraded by AmpErase UNG. If primers cannot be selected with dA nucleotides near the ends, the use of primers with 3. terminal dU nucleotide should be considered.

Submission Guidelines

  1. Sample Preparation. We recommend that users set their own reactions. However under certain circumstances we should be able to set complete reaction for an additional fee. The samples should be supplied in 96 or 384 well plate ready to be placed in the instrument. We will be responsible for running the plate on the instrument and initial data analysis. All the sample information needs to be provided in the special template format so that it could be directly imported into the SDS plate document. TEMPLATES Please open these as Excel files. Substitute your information in the fields on the template. Save the document as a text file and then import it from the text file.
  2. Plate set up. Plate set up will vary depending up on the experiment. Following are the general guidelines. For detailed protocols please contact us.
    • Each sample should be at least in duplicates. Triplicates are preferred since reaction which is out of range can be eliminated
    • NTC No template control for each RNA/DNA
    • No RT control for each cDNA. No RT control serves to determine whether there is genomic DNA contamination in the RNA preparation