Title:

Cloning and Sequencing of the GAPC gene in Spinach

Martha Holt
Melissa Huamancondor
Biology Department, Western Carolina University, Cullowhee, NC 28723

Abstract:
The main purpose of this experiment was to produce a clone portion of a GAPDH gene
(glyceraldehyde-3-phosphate dehydrogenase), from spinach, then sequencing its DNA. First,
The initial PCR indicated no band for spinach, however the bands that are visible are due to
primer dimers. Now, in the Nested PCR indicated three present bands in the middle, which
would then indicate that primers match up and can help continue the experiment.The
experiment required many methods to proceed with the results, for example gel electrophoresis
(used on initial and nested PCR, as well with digest), purification, ligation and transformation. In
the outcome of the experiment, after running the gel electrophoresis using the plasmid vector
(pJET 1.2) and BglII restriction enzyme, the visible bands were present at 1000bp and not at
3000 bp. With the DNA sequencing BLAST found a 99.8% identical sequence to Spinacia
oleracea L (spinach) sequence. This identical sequence can show that the plant is paired up
with spanish for the GAPC 1 gene.

Introduction:
The purpose of this experiment was to clone/amplify a portion of a glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene from a plant of interest, inserting this gene fragment into a
plasmid vector, transform the vector into E. coli cells, and then purify the cloned fragment from
E. coli and analyze the sequence using bioinformatics. The gene of interest was the GAPC
gene present in the Spanish. When the genomic DNA was extracted, the product was taken to
be amplified using PCR. The amplified gene fragment was inserted into a cloning vector and E.
coli cells, giving results to a cloned sequence, which can be analyzed with Geneious, a
bioinformatic program.
GAPDH genes are located in the genome of all plants, and other organisms. GAPDH genes are
considered housekeeping genes by the encoded enzymes which catalyze an important step of
glycolysis, a stage of respiration that occurs in all living cells. This project allowed us to perform
novel research, giving us a chance to clone and sequence a gene from a plant that has not yet
been analyzed.

Results:
Initial PCR Gel.
This figure demonstrates the initial PCR reaction of red maple, spinach, arabidopsis, pGAP, and
water. There are no PCR products for Red Maple or Spinach for the initial PCR reaction. The
visible “bands” of the Red Maple and Spinach are due to there being 24 primers added in
excess and it is possible that they were binding to one another. These primers hybridize to one
another when they bind producing primer dimers which are the bands that are visible in lane 2.
Arabipopsis had a band present around 1,500 bp and pGAP had a band present around 1,250
bp.

Nested PCR Gel.

This figure demonstrates the nested PCR reaction of red maple, spinach, arabidopsis, pGAP,
and water. There were no bands present for the Red Maple nested PCR reaction. The Spinach,
arabidopsis, and pGAP generated a PCR product at a size of 1,000 bp. This indicated that the
amplification of spinach DNA in the first round of PCR was successful.

Digest Gel.
This figure demonstrates the gel electrophoresis of the pJET 1.2 plasmid and the Bgl II
restriction enzyme of the four minipreps of plasmid DNA. The four minipreps contained
Escherichia coli and were mixed with DNA from Spinach. Bands were present at approximately
1,000 bp in wells one, three, and four.
Bioinformatics:

Description E-value Bit-Score % Pairwise Query Grade

Identity Coverage

Dalea 0 937.235 99.8% 100.0% 99.8%

purpurea

Pilea cadierei 0 941.743 99.8% 100.0% 99.9%

GapC gene

Arabidopsis 0 936.333 99.8% 99.43% 99.6%

Thaliana

Ageratina 0 934.53 99.8% 99.24% 99.5%

altissima

Petunia sp. 0 934.53 99.8% 99.24% 99.5%

Table 1: Presenting the top five Contig BLAST Results that was presented by Geneious,
showing the E-value, Bit-score, % Pairwise Identity, query coverage and grade. Dalea purpurea
is the closest match to our sequence, with Petunia sp being a great match, however not as
close as to Dalea Purpurea.

Gene Arabidopsis Beginning of End of Query Query Bit-Score for

Chromosome Arabidopsis Arabidopsis Begin End Query
Gene Gene Sequence Math
Location Location

GAPC 3 1,080,957 1,083,537

Query 1,082,031 1,082,551 4 524 936.33

Table 2: Homology table as determined by bioinformatic program (Geneious), showing gene

location, Query coverage, and bit-score of the GAPC and query. Query is our sequence that
was observed by Geneious, it's the gene that we wanted to clone for this experiment. It’s
organized to compare our query to an actual GAPC gene.
Figure 1: The alignment view of the Contig sequence representing the GAPC1 gene, made by
Geneious. The GAPC1 being shown is presented by our query shown in Table 2, showing the
length of the nucleotides.

Conclusion and Future Work:

In conclusion, the PCR results specify that the cloning and sequencing of the GAPC from the
Spinach was unsuccessful potentially due to human error and contamination while carrying out
the experiment procedure. The pGAP control used for DNA sequencing and bioinformatics
analysis suggested that the cloning and sequencing of GAPC from pGAP was

In the future, it would be beneficial to complete this experiment again while focusing on the Red
Maple plant rather than Spinach to be able to compare with the data collected from classmates.
To potentially replicate a more complete genome different genes from Spinacia oleracea can be
cloned and sequenced.

Acknowledgements:
We would like to thank Chistopher Beyer for all his help during this lab experience, as well as
Hannah Dinkins for her efforts. Thank you Western Carolina University for the opportunity for
this lab experience.

References
1. Harris & Beyer. Biology 333: Cell and Molecular Biology Lab Manual. 2020.
2. Harris & Beyer. Cell and Molecular Biology Gene Cloning and Sequencing Supplemental
Lab Manual. 2020.
3. Geneious Prime Bioinformatics Program.