In Chebuu/Design-Group-Specific-Primers:

knitr::opts_chunk$set(
  collapse = TRUE,
  comment = "#>"
)

library(DesignGroupSpecificPrimers)

data(THCAS_DNA)

dbconn <- RSQLite::dbConnect(RSQLite::SQLite(), ':memory:')

groups <- NULL
tiles <- NULL
identifier <- NULL

designGoupSpecificPrimers(
  xstringset = THCAS_DNA, 
  groups = groups, 
  tiles = tiles, 
  identifier = identifier, 
  dbConn = dbconn
)

Simulating the Onofri 2015 primers

NOTE:: Simulating two oligos against the entirety of chromosome 6 would take ~2wks on my desktop...

Cannabinoid synthases are located on Ch.9

Both thcas and cbdas are located on chromosome 9 () or I guess chromosome 6???? (). Use the Onofri primers to perfom in-silico PCR on Ch.9 of the of strain CBDRx.

library(Biostrings)
library(DECIPHER)

library(DesignGroupSpecificPrimers)

# Cannabis sativa chromosome 6 (NCBI GenBank)
CANSAT_CH9 <- Biostrings::readDNAStringSet(
  system.file(
    'extdata/CANSAT_CH9.fasta', 
    package = 'DesignGroupSpecificPrimers'
  )
)

# THCAS/CBDAS sequencing primers (Onofri et al., 2015)
data(Onofri2015_THCAS) # Biostrings::DNAStringSet
data(Onofri2015_CBDAS) # Biostrings::DNAStringSet

thcas_amplicons_ch9 <- DECIPHER::AmplifyDNA(
  Onofri2015_THCAS, 
  CS10_CH9, 
  annealingTemp=55, 
  P=4e-7, 
  maxProductSize=5000
)

A BBE-like domain exists on Ch.6

[According to who/what?] A BBE-like domain existst on Ch.6 that could yield off-target amplicons. Perform the same PCR simulation with Onofri primers on cs10 Ch.6.

# Cannabis sativa chromosome 6 (NCBI GenBank)
CANSAT_CH6 <- Biostrings::readDNAStringSet(
  system.file(
    'extdata/CS10_CH6.fasta', 
    package = 'DesignGroupSpecificPrimers'
  )
)

thcas_amplicons_ch6 <- DECIPHER::AmplifyDNA(
  Onofri2015_THCAS, 
  CS10_CH6, 
  annealingTemp=55, 
  P=4e-7, 
  maxProductSize=5000
)

Designing PCR Primers

MOLECULAR DIAGNOSTIC PCR HANDBOOK
ISBN-10 1-4020-3404-0(e-book)
GERRIT J. VILJOEN
International Atomic Energy Agency, Vienna, Austria
LOUIS H. NEL
University of Pretoria, Republic of South Africa
and
JOHN R. CROWTHER
International Atomic Energy Agency, Vienna, Austria

Agarose Gels

| Agarose (%) | DNA length (bp) | |---------|--------------| | 0.3% | 1000 - 30,000 | | 0.7 | 800 – 12,000 | | 1.0 | 500 – 10,000 | | 1.2 | 400 – 7,000 | | 1.3 | 300 – 3,000 | | 1.4 | 250 – 3,000 | | 1.5 | 200 – 3,000 | | 2.0 | 50 – 2,000 |

Chebuu/Design-Group-Specific-Primers documentation built on Aug. 17, 2020, 1:29 a.m.