TITLE: PCR protocol to identify an equine mutation associated with Warmblood Fragile Foal Syndrome(WFFS)
AUTHORS: gerald.barry , Sharon Flanagan, Áine Rowe, Vivienne Duggan, Erin Markle, Maureen O’Brien

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Warmblood Fragile Foal syndrome (WFFS) is an autosomal recessive condition that affects the maturation of collagen in affected foals. Foals affected with the disease typically die or are euthanised shortly after birth. WFFS is caused by a single nucleotide change at position 2032 of the equine PLOD1 gene, causing an impairment of the wild-type enzyme. A commercial test for the causative genetic mutation is currently available from companies operating under licence from Cornell University but it has limitations. This test requires amplification of a region of the PLOD1 gene encompassing the site of interest, followed by Sanger sequencing of that region and computational analysis. We present here the development of an alternative, real-time PCR based assay that rapidly and reliably differentiates between the wild-type and WFFS associated nucleotides without the need for sequencing, thus increasing the potential for high throughput analysis of large numbers of samples in a cost-effective manner. </div></div>

[STEPS]
?. [DNA extraction]
Hair strands (10 per extraction) are cut at a maximum length of 1 cm, including the follicular tag.
?. [DNA extraction]
The hair is placed into a 1.5 ml Rnase, DNase-free micro-centrifuge tube followed by 300 µl of ATL Buffer, 20 µl of Proteinase K and 20ul of a 1 M DTT solution.
?. [DNA extraction]
The sample is incubated at 56oC with occasional vortexing until the hair is completely lysed and cannot be seen anymore.
?. [DNA extraction]
After incubation, the sample is vortexed for 15 seconds to ensure complete homogenisation.
?. [DNA extraction]
300µl AL Buffer is added and the sample is again briefly vortexed.
?. [DNA extraction]
300µl of ethanol (molecular grade, > 99.5% pure) is added with brief vortexing again to ensure complete mixing.
?. [DNA extraction]
The sample mixture is pipetted into a DNeasy mini spin column and centrifuged at room temperature at 6000 x g for 1 minute. The flow through is discarded
?. [DNA extraction]
500µl of AW1 Buffer is added and the column is centrifuged at room temperature at 6000 x g for 1 minute and the flow through discarded.
?. [DNA extraction]
500µl of AW2 Buffer is added and the column is centrifuged at room temperature at 6000 x g for 1 minute and the flow through discarded.
?. [DNA extraction]
The samples are centrifuged again at room temperature at 6000 x g for 1 minute to remove any excess ethanol before the extracted DNA was eluted into a clean RNase/DNase-free tube.
?. [DNA extraction]
For elution, 100 µl of buffer AE was added to the column, allowed to incubate for 1 minute at room temperature and then centrifuged at room temperature at 6000 x g for 1 minute.
?. [DNA extraction]
The sample is re-added to the column, re-incubated for 1 minute and then centrifuged at room temperature at 6000 x g for 1 minute.
?. [RT-PCR]
PCR Step
    Temperature
    Time
    Cycles
    Pre – Read
    60oC
    30 seconds
     
  Hold
    Initial denaturation / Enzyme Inactivation
    95oC
    5 minutes
    Denature
    95oC
    15 seconds
    40
    Anneal / Extend
    60oC
    60 seconds
    Post – Read
    60oC
    30 seconds
    Hold
PCR Step
    Temperature
    Time
    Cycles
    Pre – Read
    60oC
    30 seconds
     
  Hold
    Initial denaturation / Enzyme Inactivation
    95oC
    5 minutes
    Denature
    95oC
    15 seconds
    40
    Anneal / Extend
    60oC
    60 seconds
    Post – Read
    60oC
    30 seconds
    Hold
?. [RT-PCR]
Each PCR reaction is set up as per the table below in a 96 well plate.

 Component  Volume per 20 µl reaction  TaqPath ProAmp Master Mix  10 µl  TaqMan Genotyping Assay  0.5 µl  Genomic DNA or NTC  5.0 µl  Nuclease- Free water  4.5 µl  Total Volume  20 µl
Component  Volume per 20 µl reaction  TaqPath ProAmp Master Mix  10 µl  TaqMan Genotyping Assay  0.5 µl  Genomic DNA or NTC  5.0 µl  Nuclease- Free water  4.5 µl  Total Volume  20 µl
?. [RT-PCR]
The well plate is sealed with PCR compatible optical adhesive film and centrifuged at room temperature at 220 x g for 1 minute.
?. [RT-PCR]
Primers used are as follows:  Component 
    Sequence 
    SNP Genotyping assay Forward Primer 
    TCCTGTTGGGAAACTGACACTTC
    SNP Genotyping assay Reverse Primer 
    TCGGATGGAGCAGTTGTAACG
    FAM Probe 
    ACAGCCCCTGCCCTG
    VIC Probe 
    CAGCCCCCGCCCTG
Component 
    Sequence 
    SNP Genotyping assay Forward Primer 
    TCCTGTTGGGAAACTGACACTTC
    SNP Genotyping assay Reverse Primer 
    TCGGATGGAGCAGTTGTAACG
    FAM Probe 
    ACAGCCCCTGCCCTG
    VIC Probe 
    CAGCCCCCGCCCTG