Exercise is from 52 oC to 58 oC. Runs

2: Primer Design

and Discussion

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Sequence from E. coli poIA gene

Intended Target:  Escherichia coli str. K-12 substr. MG1655


          The following shows the primers
generated using Real Time Primer-BLAST to amplify a specific DNA sequence in  Escherichia coli str. K-12 substr. MG1655:









conditions must be considered to identify the efficacy of primers before it is
use to amplify a desired and specific gene sequence. All generated primers have
a product size of 300 base pairs, which is the desired size of the sequence to
be amplified. The primer lengths of all generated primers are all 20 bases
long. This follows the optimum number of a primer length which is actually 20
bases long. All primers have GC% that is ranging from 40% to 47 %. This still
follows the ideal percentage of GC’s in a primer which should be 40%-60%. All
generated primers have GC clamp which is essential because it ensures the
specificity of the primers as it anneals to the template.

melting temperatures of the primers are ranging from 53 oC to 55 oC
which is still within the ideal melting temperature for a primer which is from
52 oC to 58 oC. Runs of bases have been observed in
primer pair 1. The reverse primers have 5 repeating T’s and this could lead to possible
mispriming. All primers have been observed to have a self-complementary scores
ranging from 3.00 to 6.00. This could indicate a low possibility that the
primers will bind to itself. The Self 3′ complementary scores of the primer
pairs are ranging from 0.00 to 3.00. This is considerably low and so there is a
low chance that a primer dimer formation will occur.

            As for specificity, all the primers have found to be
specific on their target templates. In conclusion, primer pairs 2 to 5 have met
the expected conditions to be an effective primer. The reverse primer of the
primer pair 1 have been observed to have an exceeded repeating bases of T’s
that could lead to possible mispriming.


1. The default primer sequences in the PCR simulator with
bacterial templates are ‘universal’ ribosomal RNA primers, which work on
(almost) any species of bacteria. These primers are designed to match highly
conserved regions of the rRNA sequences. Find a paper describing how
pan-species primers like these are designed and, citing the paper, explain how
these primers are made.

Materials and Methods

Fecal samples

            Fecal samples were obtained from 3
11-month-old crossbred pigs. The samples were the divided into 5-g samples and
left frozen at ?80°C.

DNA extraction

mg of each sample was treated in 4 M guanidium thiocyanate, 100 mM Tris-HCl (pH
9.0), and 40 mM EDTA after being thawed on ice. The samples were then pounded
with zirconia beads. DNA was extracted from the bead-treated suspension. The concentrations
of DNA were estimated using a spectrophometry. The final concentration of the
extracted DNA was adjusted to 10 ng/µL.

Development of a prokaryotic universal
primer set

            Published universal PCR primers sets
which target V3-V4 region of archaeal and bacterial rDNA were altered to
augment the detection rate of prokaryotes. Several alignments of 16s rDNA
sequences were obtained from a database. Based on the alignments, the sequence
of the primers with the fewest mismatches in the V3-V4 hypervariable region of
the 16 rRNA were selected as the prokaryotic universal primers.

Illumina library generation

            The region that have been amplified
in Prokaryotes, Bacteria and Archaea were the V3-V4 region. These primers were
also complementary to both forward and reverse primers of Illumina. For
multiplexing, the reverse primer has 6-bp sequence. Illumina adapters were used
to designed amplification primers.

            To eliminate the formation of
unwanted products during the process of amplification, touchdown PCR method for
thermal cycling was used together with a Rotor-Gene quantitative thermal cycler.
The mixture for the reaction contains 10
ng genomic DNA, MightyAmp for Real Time and 0.25 µM of each primer. The
conditions for PCR reaction were as follows:

denaturation at 98°C for 2 minutes,

35 cycles of
annealing beginning at 65°C and ending at 55°C for 15 sec,

extension at 68°C
for 30 sec,

temperature was lowered 1°C every cycle until reaching 55°C, which was used for
the remaining cycles.

PCR products were purified using a
MultiScreen PCRu96 filter
plate and was analyzed using a MultiScreen PCRu96 filter plate to detect any presence of
primer-dimers and to identify the average molecular weight of each product. The
purified products were quantified by real-time quantitative PCR (q-PCR) on a
Rotor-Gene Q quantitative thermal cycler using MightyAmp for Real Time , 0.2 µM
of each primer, which were derived from Illumina adapters, and serially diluted
PhiX control library as a standard.

The conditions for the PCR reaction for
quantification of each PCR product produced were as follows:

: initial
denaturation at 98°C for 2 minutes,

30 cycles of
denaturation at 98°C for 10 sec,

annealing at 60°C
for 15 sec, and

extension at 68°C
for 30 sec

The quantification was used to identify
the concentration of the amplified libraries and to confirm if there is the
presence of primers for Illumina sequencing.



Illumina sequencing and quality filtering

            Each of the multiplexed library tool
was applied with 25% phiX control to refine base calling during sequencing. A
sequencing process was done by using a paired-end, 2×250-bp cycle run on an
Illumina MiSeq sequencing system and MiSeq Reagent Nano Kit version 2 (500
Cycle) chemistry. A paired-end sequencing with read lengths of 251 bp was
performed. Reads with a quality value scores of > 20 for more than 99% of
the sequence were obtained for analysis.

16S rDNA-based taxonomic analysis

            The Ribosomal Database Project was
used to analyse the sequence of reads manually. Reads gathered in the FASTA
format were assigned to levels with an 80% of confidence threshold.

Real-time quantitative PCR

Quantitation of
all Bacteria
and Archaea in
the pig intestinal tract was conducted using real-time quantitative PCR .

Purified genomic DNA from a bacteria and the
extracted fecal DNA was amplified was amplified using primer pairs. The mixture
for the PCR reaction contains the following:

20 ng genomic DNA,

2×MightyAmp Buffer

0.25 µM of each
primer and

1.25 units of
MightyAmp DNA Polymerase

The cycling conditions were as follows:

denaturation at 98°C for 2 min,

followed by 35
cycles of 98°C for 10 s,

55°C for 15 s, and
68°C for 1 min

products for amplification were mixed with 1 µL EZ-Vision One DNA Dye and then
separated by electrophoresis. The products were then purified using Econo Spin
IIa and later cloned into pGEM-T Easy vector for the transformation of E. coli HST08 Premium Competent Cells. In the
agar treated with ampicillin, positive transformants were identified and
obtained. A colony that was identified by PCR to have  a plasmid with the expected DNA was cultured
in in 5 ml LB medium treated with ampicillin (100 µg/ml) overnight. The culture
underwent a centrifugation to pellet the cells. Plasmid was extracted from
cells using QIAprep Spin miniprep kit. Quantification of the purified plasmid
was done using a ND-1000 instrument.The number of 16S rDNA copies present in
the preparation was estimated.


Reference: Takahashi S, Tomita J, Nishioka K, Hisada T, Nishijima M
(2014) Development of a Prokaryotic Universal Primer for Simultaneous Analysis
of Bacteriaand Archaea Using Next-Generation Sequencing.
PLoS ONE 9(8): e105592. https://doi.org/10.1371/journal.pone.0105592


2. Find PCR primer sequences in bacterial species
assigned your group in scientific papers that work in the PCR simulator.
Evaluate their performance based on how you evaluated the primers you designed



            The primer pair on Fig. 6 has a shorter
primer length of 18 bases and 19 bases for the forward primer and the reverse
primer respectively. This is less than the optimum length of a primer which is
20 bases. Its melting temperature is still within the ideal range which is 52 oC and 59.77 oC.
GC% is also within the ideal range of 40% to 60% in a primer. The primer has greater
values for self-complementarity which means this primer has a higher
probability of binding to itself. The values of Self 3′ complementarity are
lower and this indicates a low probability of forming a primer dimer.


Reference: Sabat, G., Rose, P., Hickey, W., & Harkin, J.
(1999). Selective and Sensitive Method for PCR Amplification of Escherichia
coli 16S rRNA Genes in Soil. Retrieved from