Molecular cloning using polymerase chain reaction, an educational guide for

cellular engineering
Sayed Shahabuddin Hoseini1,2 and Martin G Sauer1,2,3*
Molecular cloning is used to produce more genetic material for testing. This paper explains one
of the main ways that DNA can be sequenced. DNA sequencing is a difficult thing to do and so this
paper outlines a straight forward way to achieve a good result. Bacteria and viruses and even tomato
proteins are used in cloning and help produce the needed DNA. It is interesting how this can be done
and this article shows a way to do this.
Introduction DNA sequences were cloned using PCR primers with embedded restriction
enzyme sites. There are many ways to to do DNA sequencing. The use of restriction endo-nuclease
enzymes is the most used technique. It is especially effective when compatible restriction enzyme sites
are available on both, insert and vector DNA sequences. However sites can be replicated and used even
if they are missing or damaged. Through the use of PCR kits. Even though PCR cloning has been used
for a long time, the procedure is not easily found or widely accessible. PCR cloning is fast and
efficient thanks to DNA polymerases, kits, and powerful new software. The main reason for this article
is explaining the sequential process of PCR cloning.
Method Cell lines and media E. coli was used for preparation of the cloning. Human embryonic
kidney cells were cultured and injected. Plasmids, primers, PCR and sequencing plasmids provided by
Axel Schambach primers dissolved with ultra pure water. The PCR using a peqSTAR thermocycler
Sequencing used clonejet PCR kit manipulation of DNA fragments to view plasmid maps. Then clone
software used to analyze the results. DNA was mixed with dye and run in a gel, then a litigation
calculator was designed and used for easy calculation of the required insert and vector volumes. DNA
was isolated and used Production of viral supernatant and transduction of cells. The cells were thawed
and grown. Transfection mixtures were made virus were harvested and the results were read through
flow cytometry and fluorescence microscope viewing.

Results and Discussion Choosing proper restriction enzymes was based on a defined criteria.
An example was, putting tdTomato fluorescent protein in Murine leukemia cells to track tumors in
mice. This process will work on any number of clone able proteins. First the star codon and stop codon
are found. The tdTomato gene has ATG start codon and TAA stop codon. Some times a start spot or
stop spot must be added. The cloning product were then screened for the right orientation, reverse
orientation, or self-litigation. Sticky end cutters were the most effective way to cut DNA, and blunt end
cutters were also be used f to cut the DNA sequences at the correct sites. It was important to make sure
that only the correct spots are cut so that the DNA sequences are usable for cloning. Some methelated
DNA needed to be prepped in E. coli because with out the E coli they can resist cutting by
methylation-sensitive enzymes. Cloning is easier if the buffer works for both full functionality of
restriction enzymes. This is possible with increases in concentration. This allowed for less loss of DNA
during purification.
Designing cloning primers based on defined criteria. For the PCR primers design, the start and
stop codons were check and a Kozak sequence (GCCACC) was used before the ATG. Inserting Kozak
sequence before ATG improved translation. The temperature was calculated for the reverse primer and
forward primer. Performing PCR using proofreading polymerases. The PCR reactions followed
logarithmic amplifications, because this increased the results any errors were also amplified. It is
important to know the error rate so that DNA is with in acceptable ranges. The DNA is loaded into a
gel and the extracted PCR was sequenced using cloning kits. A toxic gene can be used to control
bacteria growth that could cause false readings in the DNA sequences. The colonies that survived are
the colonies that have no errors.
Analysis of sequencing data. Sequencing companies report data as a FASTA file and as ready
nucleotide sequences. For sequence analysis, the following websites were used:
http://blast.ncbi.nlm.nih.gov/Blast.cgi, http://xylian.igh.cnrs.fr/bin/align-guess.cgi these websites help

show sequences and provide alignment data. Nucleotides should match up 100% but there are error
rates that all sequencing processes have these if the identity is not 100% the PCR should be rerun. The
normal read lengths are around 800 to 900 topping at 1800dp for Sanger sequencing. DNA clones were
removed and checked for purity. Viral production and transduction of target cells was through the use
of kidney cells used for easy culturing. Murine Leukemia cells were annualized through the
fluorescence of tdTomato. The leukemic cells were transduced with freshly harvested virus. The virus
showed the presence in tdTomato in the majority of the cells.
Conclusion PCR cloning was explained through the cloning of DNA and the use of one PCR
process. Some alternative PCR processes exist that were not covered in this article. This gathered
information is to help minimizes errors and pitfalls for future PCR cloning so that testing will be more
efficient in the future.