Why Align Sequences

Why Align Sequences

General Information

"Although primer mismatching does not necessarily prevent PCR amplification, use of primers having a known high degree of sequence conservation...should reduce assay variability in clinical practice" (Chou 1992 J of Clin Micro 30(9): 2307-2310).

"A day in the library saves a month in the lab."

• Primer size and location of mismatch affect amplification efficiency which can effect clinical sensitivity and make defining a clinical threshold for treatment difficult.



• Ensuring primers and probes sit on locations with high degree of conservation is a quick, easy and cheap way of deciding if the assay is worth the time and effort involved in empirical testing of clinical samples.

Examples

When performing statistical analysis after empirical testing with clinical samples your primary concern should be "what should be considered the clinical threshold for treatment?" Not "will this assay work well enough for diagnostic use?" When a PCR assay is designed on a highly polymorphic sequence – like in example 1 – some strains will yield false negative results. On the other hand example 2 shows a PCR assay on a well conserved sequence. And the author spends time determining the appropriate threshold for treatment.

• The core of our sequence alignments consist of eight CMV strains with complete genome submissions found in Genbank



• Partial sequences will be added to alignments when informational value is added



• Alignments will allow researches to search for primers to quickly determine the degree of sequence conservation

Example 1

We have taken 14 sequences for different CMV strains and aligned them in the UL122 region (major immediate early), marked the locations of one set of primers and probe and edited out the intervening sequence since it does not impact the assay.



For high resolution image click here

The above qPCR assay:

• Published Tanaka 2000 Journal of Medical Virology 60:455–462



• Targets the UL122 region



• Has a false negative rate of 24% (Tanaka 2000)



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  • There are 8 unique locations where a mismatch occurs
  
  
  
  • 7 mismatches are found in the Toledo strain (AC146905)
  
  
  
  • Only AD169, Towne, Merlin and one clinical isolate (4/14) do not contain a mismatch with this qPCR assay
  
  
  
  • Future researchers can avoid spending time and money testing assay which have a high degree of sequence heterogeneity
  
  
  
  

Example 2

We took our eight core sequences, aligned them in the US17 region and mapped the locations of one primer and probe set.

For high resolution image click here

The above qPCR assay:

• Published Sanghavi 2008 Journal of Clinical Virology 42: 335-342



• Targets the US17 region



• Authors spent time determining - via statistics - the viral load which should be considered the threshold for treatment



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  • There is only one observed mismatch at the 5' end of the reverse primer