Methods and Techniques in Molecular Biology

Running head:TECHNOLOGY OF MOLECULAR BIOLOGY
TECHNOLOGY OF MOLECULAR BIOLOGY
Name of the student
Name of the university
Author note

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Part I
Answer 1.
A and B are identified as a third-generation sequencer, or long-read sequencer and C
is identified as short read sequencer. Long read sequencing is more advantageous than short-
range sequencing. Pacific Biosciences’ (PacBio) single-molecule real-time (SMRT)
sequencing and Oxford Nanopore Technologies’ (ONT) nanopore sequencing are two
important technologies that dominate long-read sequencing. These technologies provide
reads more than 10kb. The data of third-generation sequencing highly differs from second-
generation sequencing. Illumina’s Novaseq, HiSeq and MiSeq instrument produce short-read
sequences that produce up to 600 bases. Short read sequencing is precise, worthy and assisted
by a variety of tools that helps in analysis.
SMRT sequencers detect fluorescence related events that tie in with an inclusion of one
particular nucleotide with the help of polymerase which is fastened to the base of tiny well
whereas nanopore sequencers estimate the ionic current fluctuations when a single-stranded
nucleic acid move through biological nanopores (Nakano et al. 2017). The full-length
transcripts are produced by Iso-Seq method using SMRT sequencing. The Nanopore
sequencing from 500 bp to 2.3 Mb along with 10-30 kb genomic libraries provides the
longest read length (Byrne et al. 2017). In SMRT sequencing, the read length is restricted by
the longevity of the polymerase. The library inserts size range from 250bp to 50kbp. Whole
Genome Sequencing, Epigenetic, targeted sequencing, RNA sequencing and Complex
Population can be done with SMRT Sequencing with high accuracy. On the other hand, in
short-read sequencing natural nucleic acid polymer span eight orders of magnitude in length
and sequence them into small amplified fragments which complicate the task of rebuilding
and evaluating the original molecules whereas the long-read sequencing improves mapping

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certainty, detection of structural variants, de novo assembly and transcript isoform
identification (Shi et al. 2017).
The schematic diagram is identified as whole-genome sequencing. The whole-genome
sequencing is considered as the analysis of the entire genomic sequence of a cell at a time,
thus provides a comprehensive characterization of the genome. This process of whole-
genome sequencing will help in detecting single nucleotide variants, large structural variants,
and change in copy number, insertions and deletions (Belkadi et al. 2015). This information
will help to obtain inherited disorders, tracking inherited disorders. The purpose of
conducting a whole genome sequence is it provides a high-resolution and screened base-by-
base. It has the potential to capture both large and small variants and detects potential
causative variants for further study of gene expression and regulation mechanisms.
Additionally, it provides a large volume of data in a short period. The large whole-genome
sequencing provides information for population genetics and is highly used to research any
disease. The small whole-genome sequencing is useful to do molecular epidemiology studies,
environmental metagenomics and infectious disease surveillance. The scaffolds gives data on
the long-range of genomic structure instead of indicating the actual DNA sequence that is
present within the gaps in middle of the contigs. The scaffolding process provides
information on the relative position of genomic segments along the chromosome. The
histogram from Illumina will provide the information on the separations in middle of natively
generated paired-end reads (Gökkaya 2020), the length of reads is generated by PacBio and
Oxford Nanopore technology and the optical maps provide information on the lengths of the
fragments which is mapped by BioNano nanocoding technology (Mikheyev and Tin 2014).
From the alignment of the contigs, the order and orientation are inferred. The long reads line
up to the terminal side of the contig implies their adjacency, and the optical maps infer the

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orientation and order of the contigs by lining up the inferred restriction pattern to that of the
experimental map. The long sequencing reads are the cases of subcloning.
Answer 2.
Optical mapping is an approach that is used primarily to get a physical map of a
genome through the construction of particular fingerprints left by large labelled DNA
molecules. This technique is used for constructing Optical mapping is an important and
powerful tool that is used to interpret the function and structures of genomes. This approach
captures a high level of a complex genomic sequence, producing a scaffold for renewed
interpretation of sequencing data of a specific level. They are specifically useful for
scaffolding de novo sequences and for discovering structural repetitions and variations within
complicated genomes. Contrasting and Comparing individual optical maps will help to focus
on the structural variations and sequence copy number variation along with giving positional
information. An accessible technology named optical mapping is utilized for validation of
genome assembly. The flowchart of genome assembly can be categorised into two-phase:
Sequence assembly and genome assembly. The sequence assembly consists of sequence data
from mate pairs, short reads and long reads to produce contigs and scaffolds. The genome
assembly phase consists of extra information such as genetic maps, prior assemblies or Hi-C
read pairs to orient the contigs into pseudomolecules, which are the representation of the
chromosomes. The advantage of optical mapping is it requires a minimal amount of DNA
sample. The optical mapping has been very useful for whole-genome assembly. It is possible
to obtain an assembly of the complete genome through the overlap of restriction enzyme
fragment maps. The use of two or more restriction enzymes to produce independent optical
maps for the same genome gives more information and thus gives more accurate result.
However, genome assembly is time taking but overcome the issues that come up due to short-

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read technologies. Optical mapping reinforces the assembly of the genome from diploid
organisms having multiple chromosomes (Tang, Lyons and Town 2015).
Part II
Answer 3
To clone EPSPS-1 gene into the MCS region of pET20a vector, we need to insert the
sequence of the gene after the His-Tag (147…157 bp). This is because the gene which is
cloned here requires an N-terminal His tag in its protein. This is only possible when a His-tag
is present before the start of a protein sequence and in turn the gene sequence.
Vector image:
Fig 1: PET20a vector
Source: Clone manager V.10
List of available enzymes: NcoI, BamHI, EcoRI, SacI and SalI.
SalI will be used to cut the cloning vector first since it is the only enzyme available which has
its cutting region near the His-tagged region.

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The EPSPS-1 gene will be inserted in between the cut ends, and the vector will be ligated.
Sequence of the specific area after the insertion (to be primed):
5’-gtggtggtggtggtggtgcttcttcaatcta…………………………………………………
ccgaattcgatgtcgagggactccaggcttgc-3’
Black colour denotes the target gene inserted between the His-tag (left red), and SalI cut
site (right red).
Primer generation:
Forward: 3’- caccaccaccaccaccacgaa-5’
Reverse: 5’-cagctccctgaggtccgaacg-3’
Answer 4.
Molecular cloning is the main component of the molecular biology to produce
recombinant DNA molecules and thus directing their replication. A cloning vector is a small
stretch of DNA that is present in an organism and foreign DNA is incorporated into for
cloning purpose. The features of multiple cloning site include the origin of replication (ori),
multiple cloning site(MCS) having restriction sites, a selectable marker and a reporter gene
(Xu et al. 2020). The cloning vector is pET- 30a(+) in which EPSPS-1 gene is inserted into
the vector’s multiple cloning site. pET-30a(+) is a bacterial vector that is used in the
expression of N-terminally S-tagged proteins. This vector has an enterokinase site, and the
reading frame that is used is pET-30c (+). EPSPS gene is involved in the biosynthesis of
aromatic amino acids such as tyrosine, tryptophan and phenylalanine.
The workflow includes as follows-
1. The specific cloning process followed to insert the EPSPS gene inside Pet-30c vector in
this study begins with cutting the vector at a HIS-tagged site so that the expression of the
insert can be checked.

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2. SalI will be used to cut the vector by using clone manager tool in order to insert the
EPSPS-1 gene inside the vector. Both the vector and the insert will be cut with the same
restriction enzyme in order to make the ligation process possible.
3. Now, genome sequencing will be done to check whether the insert has been incorporated
into the vector or not- EPSPS gene sequence inside the vector sequence will be spotted.
4. A forward primer and a reverse primer against the ESPS-1 gene sequence was generated
with primer 3 plus.
5. The primers were inserted along with the vector+insert into an E.coli host cell and
transformation will be carried out.
6. EPSPS-1 gene secretion will be identified by the synthesis of protein with HIS tag.

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Answer to the comments:
Dear student, we went through the comments as well as our file which we sent you. We
cannot proceed with the rework due to the following reasons which needs to be clarified from
your side-
1. "1. Answers to Q1 and Q2 are basically mixed together, the student mentioned everything
including optical mapping (the focus on Q2) in Q1, and then ‘mate pairs, short reads and long
reads to produce contigs and scaffolds’ in Q2. In the end, the student still did not really
propose the rationale of using several platforms together in Q1, nor suggest how optical map
information and sequence information could be integrated in Q2; "- answer 1 and answer 2
has been given differently. The question was just to identify A,B and C and then produce
rationales which we did. However, these subjective statements such as contigs scaffolds were
not mentioned to do specifically in the answer at the initial statge or in the question. The
rationales are also given after most of the sentences by supporting them with secondary
sources.
2. "he student cited many sources (e.g. XYZ, 2020) but without really providing any of the
references. The answers are unstructured and contained lots of irrelevant information, e.g.
even ‘molecular epidemiology studies, environmental metagenomics and infectious disease
surveillance’ are mentioned when the question simply asked about genome assembly in Q1;
"- The first comment is compeletely wrong as if the professor may have skipped the reference
list given at the last page of the assignement. There was no such "molecular epidemiology
studies, environmental metagenomics and infectious disease surveillance’" in our assignment.
3. "The student did not really understand the question. He did not understand the strategies in
designing primers and failed to design the primer pair. I have no choice to give him a mark of
'0' for question 1"- No explanation for detailed steps were asked in the question for the primer
design, thus we did not given any detailed writing on the primer design process.
4. Question 1 above, describe the steps of cloning the EPSPS-1 gene into the pET30a
vector.- rework has been done on this

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References
Belkadi, A., Bolze, A., Itan, Y., Cobat, A., Vincent, Q.B., Antipenko, A., Shang, L., Boisson,
B., Casanova, J.L. and Abel, L., 2015. Whole-genome sequencing is more powerful
than whole-exome sequencing for detecting exome variants. Proceedings of the
National Academy of Sciences, 112(17), pp.5473-5478.
Byrne, A., Beaudin, A.E., Olsen, H.E., Jain, M., Cole, C., Palmer, T., DuBois, R.M.,
Forsberg, E.C., Akeson, M. and Vollmers, C., 2017. Nanopore long-read RNAseq
reveals widespread transcriptional variation among the surface receptors of individual
B cells. Nature communications, 8(1), pp.1-11.
Gökkaya, A.Ş., 2020. Distributed stream-processing framework for graph-based sequence
alignment (Doctoral dissertation, Bilkent University).
Mikheyev, A.S. and Tin, M.M., 2014. A first look at the Oxford Nanopore MinION
sequencer. Molecular ecology resources, 14(6), pp.1097-1102.
Nakano, K., Shiroma, A., Shimoji, M., Tamotsu, H., Ashimine, N., Ohki, S., Shinzato, M.,
Minami, M., Nakanishi, T., Teruya, K. and Satou, K., 2017. Advantages of genome
sequencing by long-read sequencer using SMRT technology in medical area. Human
Cell, 30(3), pp.149-161.
Shi, L., Guo, Y., Dong, C., Huddleston, J., Yang, H., Han, X., Fu, A., Li, Q., Li, N., Gong, S.
and Lintner, K.E., 2016. Long-read sequencing and de novo assembly of a Chinese
genome. Nature communications, 7, p.12065.
Tang, H., Lyons, E. and Town, C.D., 2015. Optical mapping in plant comparative
genomics. GigaScience, 4(1), pp.s13742-015.
Xu, L., Chen, Y., Li, Q., He, T. and Chen, X., 2020. Molecular cloning. Fish & shellfish
immunology, 98, pp.981-987.

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