Republic of the Philippines

Cagayan State University – Andrews Campus

College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

Lesson 18
DNA TECHNOLOGY
Learning Outcomes
At the end of this chapter, students will be able to:
1. determine the importance of knowing the human genome sequence;
2. analyze DNA sequence through sequence alignment;
3. use BLAST in finding similarities with other organisms;
4. design a primer for gene amplification;
5. perform ligation of gene of interest to the plasmid; and
6. perform bacterial transformation.
Outline of Discussion
1. DNA sequence analysis
a. Genome annotation levels
b. Nucleotide-level annotation tools
c. BLAST
2. Recombinant DNA technology
a. Amplification/isolation of the desired gene of interest using a well-designed primer
b. Digestion and ligation of the gene in the plasmid
c. Transformation of the bacteria to have the recombinant DNA

DNA/NUCLEIC ACID AND SEQUENCE ANALYSIS

Nucleic acid sequencing (partial or whole genome of hundreds of species has provided re amount
of data for analysis and compartion that gave me to genomics and advancements biotechnology
and medicine The completion of the Human Genome Project (HGP) in 2003 has led to the
advancement of genetics and medicine and other related sciences (National Human Genome
Research button, n.d). HGP has allowed researchers to understand the blueprint of humans, and
has a major impact on medicine because having human genome sequence elucidates genes that
contribute to the risks of diseases, thereby leading to personalized medicine .

GENOME ANNOTATION LEVELS

Interpreting DNA/nucleic acid sequences is called DNA annotation. DNA annotation or

genome annotation is the process of identifying functional elements along the sequence of a
genome, thus giving meaning to it by identifying a possible function (Abril & Castellano, 2019),

Genome annotation includes computational annotation of long protein-coding genes on

single genomes one per species, and the experimental annotation of short regulatory elements on
a small number of them, into the population annotation of sole nucleotides on thousands of
individual genomes (many per species) (Abril & Castellano, 2019).
 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

Genome annotation has three categories :

a.Nucleotide level annotation

It identifies the physical location of DNA sequences to determine where components, such
as genes, RNAs, and repetitive elements, are located. This is the basic level of genome annotation.
However, caution mun be made at this level of annotation because sequencing and/or assembly
errors at this stage can result in false pseudogenes through indels (de Sa et al., 2018) This
nucleotide-level annotation is important in genomics.
b. Protein-level annotation
This level of annotation tries to determine the function of each gene by identifying its
protein product and its function. Moreover, the conserved regions, as well as possible amino acid
residues, interaction, and possible protein's ID structures, are discussed (Causier & Davies, 2002).
c. Process-level annotation
The goal of the process-level annotation is to identify the pathways and processes in which
different genes interact, assembling an efficient functional annotation.

NUCLEOTIDE-LEVEL ANNOTATION TOOLS

There are several bioinformatics software’s/tools available online to analyze nucleotide sequences.

a. Genome Browser

These browsers have a graphical user interface to allow for the interactive viewing of
genomic data. These are free for public access.Each software has various tools to search, view,
combine, and analyze genomic data. The genome browser gives the location of known genes, and
their functions. It combines various genome data to determine SNPs, ESTs, and conserved
sequence patterns. It allows genes to be sorted according to similar expression profiles. Moreover,
uploading own data for a specific gene and reference organism to be shared with other users is
allowed in this tool Examples of genome browsers are as follows:

• UCSC Genome Browser (https://genome.ucsc.edu/)

 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

This is created by the University of California, Santa Cruz (USA), as part of the
International Human Genome Project consortium that completed the first working draft of the
human genome assembly for public access. The website also has a genome collection of
various vertebrates and model organism assemblies and annotations for viewing, analyzing,
and downloading data It includes coronavirus data, and the tools Variant Annotation Integrator,
Table Browser, and BLAT (Genome Bioinformatics Group-UCSC Genomic Institute, 2020) .

Figure 1. The UCSC Genome Browser interface (Genome Bioinformatics Group-UCSC

Genomic Institute, 2020)

The University of California-Sta Cruz Genomic Institute created the UCSC Genome Browser to
make the human genome and other genomes accessible to the public (Genome Bioinformatics
Group-UCSC Genomic Institute, 2020),

BLAST

The Basic Local Alignment Search Tool (BLAST) is a computational tool that finds
the regions of similarity between biological sequences. The program compares nucleotide
or protein sequences to sequence databases, and calculates the statistical significance.
Figure 23 shows the BLAST interface (Altschul et al., 1990; Boratyn et al., 2012, 2019;
Madden et al., 1996; National Center for Biotechnology Institute, 2020a).

Figure 2. The interface of BLAST, a public domain created by NCBI (National

Center for Biotechnology Institute, 2020a).

Sequence similarity searches using BLAST can identify "homologous" genes or

proteins by detecting excess similarity-statistically significant similarity that reflects
common ancestry based on the available materials in the database of NCBI (Pearson,
2013). BLAST performs Pairwise Sequence Alignment to determine the regions of
similarity. Moreover, the following terms are important in analyzing BLAST results
(Altschul et al., 1990; Boratyn et al., 2012, 2019; Madden et al., 1996; National Center for
Biotechnology Institute, 2020a):
• Homology
It refers to a similarity attributed to descent from a common ancestor. Homologous
sequences have the same function; thus, new sequences can be reliably assigned functions
if homologous sequences with known functions can be identified. This homology can be
inferred based on statistically significant sequence similarity (Pevsner, 2009).
 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

• Similarity
It refers to the extent to which nucleotide or protein sequences are related. It is
based upon identity plus conservation. Furthermore, very high similarities (70%-99%)
means the sequences can be homologous sequences, but the most important indication of
homologous genes is similarity in function (Pevsner, 2009).
• Identity
It refers to the extent to which two (nucleotide or amino acid) sequences are
invariant. Identity percentage, specifically 100%, during the pairwise alignment in BLAST
is an indication that the two sequences are identical. Percent identity refers to the ration of
the number of matching residues to the total length of the alignment (e.g., 8/10 = 80%
identity! (Altschul et al., 1990; Boraryn et al., 2012, 2019; Madden et al., 1996; National
Center for Biotechnology Institute, 2020a; Pevsner, 2009).

• Conservation

It refers to the changes at a specific position of an amino acid (less commonly,

DNA) or sequence that preserves the physico-chemical properties of the original residue.
Conserved regions refer to the amino acid residues coded by DNA sequences that are found
across species, which indicate importance in terms of functions. The study of the conserved
regions across species has led to the classification of organisms/genes into the following:

a. Orthologous genes - These are homologous sequences in different species that arose
from a common ancestral gene during speciation; these may or may not be responsible for
a similar function; examples are cattle myoglobin gene and human myoglobin gene
(Pevsner, 2015).

b. Paralogous genes - These are homologous sequences within a single species that arose
by gene duplication; examples are human myoglobin gene and human alpha globin gene
(Pevsner, 2015).

RECOMBINANT DNA TECHNOLOGY

Recombinant DNA is a technology that involves inserting a human gene into the genetic
material of a common bacterium through a vector, the plasmid. Through this biotechnology,
the "recombinant organism can produce the protein encoded by the human gene (Khan et
al., 2016).

The application of this technology includes the production of insulin by inserting the
insulin gene in a plasmid placed in E. coli (shown in Figure 3). The transformed bacteria
are placed in a fermentation tank. The recombinant bacteria use the gene to begin producing
human insulin, and the scientist harvests the insulin (Khan et al., 2016), as shown in Figure
3.

Figure 3. Recombinant DNA technology steps.

 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

The steps involve restriction enzyme and DNA ligase.

Recombinant DNA technology is divided into three basic steps:

1. Amplification/isolation of the desired gene of interest using a well-designed primer

2. Digestion of original plasmid and ligation of the gene of interest to the
vector/plasmid
3. Transformation of the bacteria to have the recombinant DNA

AMPLIFICATION/ISOLATION OF THE DESIRED GENE OF INTEREST USING A

WELL-DESIGNED PRIMER

Recombinant DNA technology involves isolation of the desired gene from a human and its
insertion to E. coli. The isolation of the desired gene through gene amplification from the genomic
DNA is the first step in this biotechnology, Gene amplification/gene isolation requires polymerase
chain reaction (PCR). For PCR to be efficient, there should be well-designed DNA primers. The
primers play an important role in the amplification of the correct and complete gene (Viljoen, Nel,
& Crowther, 2005); thus, designing or using the right primer will be discussed first in this
recombinant DNA technology.

PCR primers are specific short strings (18-30 bp) of single-stranded DNA (ssDNA), known
as oligodeoxyribonucleotides or oligomers, and these flank opposite strands on either end of the
target DNA (Viljoen et al., 2005). Good primers should not have matches to other targets in certain.
orientations and within certain distances that allow undesired amplification (Ye et al., 2012).

Simplified steps in choosing a primer for your PCR mix:

Check literature and databases for existing
primers of the chosen target sequence

If NO primers are found in literature, design

primers using software and consider the parameters below.

Check the primer product should produce

DNA sequence with positive BLAST result, and its
product length should be the desired target length.

The following are the things to consider in designing a primer:

1. Primer specificity
To find the specific primer pairs, at least one primer (for a given primer pair) should
be located in regions where the PCR template does not share a high similarity to
unintended targets if possible (Ye et al., 2012).
Primer dimers, the undesired PCR product, are most often observed when one or
both of the primers bind inefficiently to the target DNA (e.g., due to the secondary
structure of the target or weak thermodynamics). When the primer dimers are
sequenced, there are often a few extra nucleotides of mysterious origin in the center
of the dimer amplicon. Primer dimers increase markedly when heterologous
genomic DNA is added (Yuryev, 2007),
2. Primer length
The primer length should not be too short because it would have low specificity,
resulting in non-specific amplification, to neither be too long that it would decrease
the template-binding efficiency at a normal annealing temperature due to the higher
probability of forming secondary structures such as hairpins. Primer length is
typically 18-30 nucleotides (Viljoen et al., 2005).
 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

3. Nucleic acid content

The G+C composition should be similar to that of the desired amplicon and
typically berween 50% and 60%. Avoid runs of three or more Cs and Gs at 3 ends
because it promotes mispriming in G/C-rich regions and avoid an A and, especially,
a T at the 3' end of a primer to allow "breathing" in the hybridization of the primer
to the template (Viljoen et al., 2005). Avoid complementarity in the 3' ends of the
primer pairs because it would lead to primer-dimer artifacts. Also, avoid any
potential mismatches in the 3' end of primers and any significant secondary
structure within primers such as internal palindromic sequences (Viljoen et al.,
2005).
4. Melting temperature or Tm
The primer melting temperature (Tm) is the most important factor in determining
the optimal PCR annealing temperature (Ta). The calculated Tm for a primer pair
should be balanced. The preferred Tm temperature is between 55°C and 72°C, and
the ideal temperature is between 62 °C and 65°C (Viljoen et al., 2005). Use the
following to calculate the melting temperature: Tm 2(A+T)+4(G+C) and -1.5°C for
every mismatch (Viljoen et al., 2005).

DIGESTION AND LIGATION OF THE GENE OF INTEREST IN THE PLASMID

The insertion of the gene of interest in the plasmid through the digestion of vector plasmid
using restriction enzyme and ligation of the gene of interest (cutting and pasting of gene) is the
next step of recombinant DNA technology (Khan et al., 2016). This is done through an enzyme
called ligase. The plasmid contains sticky ends, and the phosphatase treatment of the cloning vector
prevents self-ligation.
a. Restriction enzyme

Restriction enzymes are endonucleases that recognize short DNA sequences and
cleave double-stranded DNA at specific sites within or adjacent to the recognition
sequences (Bloch & Grossmann, 2001, Examples of common restriction enzymes are as
follows: The restriction enzymes Bam H1 and EcoR1 recognize and cut DNA at a very
specific pattern (shown in Figure 25 and Table 5) that produces ends with single-stranded
DNA "overhangs" also known as "sticky ends" that are very useful in producing
recombinant DNA. EcoR1 cutting site is shown in Figure 4 and Table 1.

Restriction endonuclease Sau3A cleaves DNA that cuts straight down at the middle of a target
sequence, thus, it is known as "blunt cutter," which is harder to ligate (blunt ends) compared to
sequences with "sticky ends" (Cruz et al., 1984).
 Republic of the Philippines
Cagayan State University – Andrews Campus
College of Allied Health Sciences
Academic Year 2024 -2025
Bagay Road, Caritan Sur, Tuguegarao City, Philippines 3500

Table 1. List of Restriction Endonuclease

Name of Restriction Source of Recognition Site Cutting Site
Enzymes Restriction Enzyme
Bam H1 Bacillus 5’ – GGATCC-3’ 5’ – G↓GATCC-3’
amyloliquefaciens 3’ -CCTAGG-3’ 3’ -CCTAG↑G-3’
Sau3A Staphylococcus 5’-GATC-3’ 5’-↓GATC-3’
aureus 3’- CTAG-5’ 3’- CTAG↑-5’
EcoR1 Escherichia coli 5’-GAATTC-3’ 5’-G↓AATTC-3’
3’-CTTAAG-3’ 3’-CTTAA↑G-3’
(Source :Cruz et al., 1984)

Figure 5. Ligation of the target gene or gene of interest

TRANSFORMATION OF THE BACTERIA TO HAVE THE RECOMBINANT DNA

Transformation is the process that involves the uptake of the naked DNA from the
surrounding medium by competent cells. Another method is through electroporation, in which
electrical pulses are introduced to washed cells, creating temporary pores that allow the entry of
DNA molecules (Werts & Low, 2017).