RNA SEQUENCE ANALYSIS AND

PCR PRIMER
By Steven Crane
 GENE NAME AND ITS PROPERTIES

• Standard Gene Name: YOR1

• Systemic Gene Name: YGR281W
• Function of Gene Product: Plasma membrane ATP-binding cassette (ABC)
transporter; multidrug transporter mediates export of many different organic
anions including oligomycin; homolog of human cystic fibrosis transmembrane
receptor (CFTR) 1 2 3 4 5 6
• Protein Size: 1477 amino acids
• Gene Size: 4434 base pairs
• Introns: No.
 YOR1 transcript levels vary upon Met4
hyperactivation in “WT” and met31 delete cells
but exhibit no change in the met32 delete cells

Transcriptional microarray heatmap profiles of YOR1 upon Met4

hyperactivation in “WT”, met31 delete, and met32 delete cells

Red: Increased YOR1 Green: Decreased YOR1 Black: No change in YOR1

transcripts compared to 0 transcripts compared to transcripts compared to 0
min sample 0 min sample min sample
 Mapped RNA-Seq reads for YOR1 from
control and promoter swap strains
315= met32 delete 793= met32 delete with MET31 driven
(CONTROL) by MET32 promoter (promoterswap)
315-1, 2, 3
793-1, 2, 3

YOR1
NORMALIZED READS OF YOR1
 Regular PCR primer set for YOR1

YOR1 PCR F: TTACCGGCAGGCGATATGAC

YOR1 PCR R: TGCATACAACGGTAGTGCGA

• “GC clamps”: both primer have GC rich 3’ends (but F primer ends in a C)
• Complementarity: much lower than 6.0
• Matched Tm’s
• At least 50% GC rich
 Quantitative PCR primer set for YOR1

YOR1 qPCR F: AGGTACTGATGGCCAAGTCG

YOR1 qPCR R: GCAACAACCGCTTCCTGTTC

• “GC clamps”: both primer have GC rich 3’ends (but R primer ends in a C)
• Complementarity: 6 and below
• Matched Tm’s
• At least 50% GC rich
 Position of Regular PCR Primers and qPCR Primers for YOR1 With PCR
Product Sizes
Size of Regular PCR product is 621

Forward Primer for Regular Reverse Primer for Regular

PCR PCR
2083-2102 2703-2684
1 4434
YOR1

Forward Primer for qPCR Reverse Primer for qPCR

2343-2362 2474-2455

Size of qPCR Product is 132