Results

1
Results
2
Base Line Standard Curve
The point at which fluororescence crosses the
threshold is CT.
In the real- time PCR, SYBR green (a
fluororescence dye) binds to the target gene.
The fluorescence increases as the dye bind to
increasing amount of target gene in a reaction
tube.
3
Melting Curve

• After real-time PCR amplification, a melting
  curve step was performed. This step is used as a
  control check to make sure that all samples have
  similar melting temperature.

4
Standard Curve

• CT values for the dilution were plotted against
  concentration. Correlation Coefficient (0.94)
  estimated is excellent.
• Gene copy numbers were then estimated in the
  samples.

5
Target gene copies
16S rDNA gene copies ranged between 1.18 x 1010
to 1.34 x 1011 genes per ml of aquifer Dehalococc
oides 16S rRNA gene copies varied between 8.8 x
10-1 and 1.19 x 1011 genes per ml of aquifer
6
Target gene copies

• qPCR results showed an abundance of
  Dehalococcoides in all aquifer samples examined,
  with D-1156 being the most abundant and D-1245
  the least abundant.

7
Dehalococcoides 16S rRNA Tree
Analysis of the sequences revealed identity to
Dehaloccoides sequences in the sequence
databases. Comparisons of 16S rDNA
Dehalococcoides sequences showed that the Dover
aquifer samples were dominated by gene sequences
related to Dehalococcoides sp. CBDB1,
Dehalococcoides ethenogenes and several
uncultured Dehalococcoides spp. several
uncultured Dehalococcoides spp.
8
BLAST Search Results
Sequence analysis of PCR assay amplicons from
Dover aquifer samples confirmed that the assay
products were Dehalococcoides with the exception
of a few sequences, which displayed closed
similarity (99) to Thermococcales archaeon and
uncultured Thermoproteales archeon (Table 1). A
total of 123 different complete sequences from
five different sites, of which 119 sequences
tested positive for Dehalococcoides.