Central dogma of molecular biology

DNA Replication
Transcription
Translation

A- aminoacyl site
P- Peptidyl site
E- Exit site
Polymerase chain reaction
DNA extraction
RNA extraction
Protein Extraction
UV-Vis Spectrophotometry

In UV-Vis spectroscopy, light is
passed through a sample at a
specific wavelength in the UV or
visible spectrum. If the sample
absorbs some of the light, not all of
the light will be pass through, or be
transmitted.
Agarose gel electrophoresis
Higher percentage agarose gels
resolve smaller fragments

Lower percentage agarose gels

resolve bigger fragments
Southern Blot Northern Blot
Polyacrylamide gel electrophoresis
Western Blot
DNA Sequencing Technologies

• Sanger sequencing
• Capillary electrophoresis and fragment analysis
• Next-generation sequencing
Sanger sequencing
Capillary electrophoresis and fragment analysis
Capillary electrophoresis (CE) instruments are capable
of performing both Sanger sequencing and fragment
analysis. Fragment analysis is a method in which DNA
fragments are fluorescently labeled, separated by CE,
and sized by comparison to an internal standard. While
DNA sequencing by CE is used to determine the
specific base sequence of a particular fragment or gene
segment, this technique can also provide sizing,
relative quantitation, and genotyping information for
fluorescently labeled DNA fragments produced by
PCR using primers designed for a specific DNA target.
Next-generation sequencing
The spectrum of analysis of NGS can extend from a small
number of genes to an entire genome, depending on the goal.
Whole-genome sequencing (WGS) and whole-exome sequencing
(WES) provide the sequence of DNA bases across the genome
and exome, respectively. Whole-transcriptome sequencing
provides sequence information about coding and multiple
noncoding forms of RNA to assess variations and gene
expression levels across the entire transcriptome. Targeted
sequencing covers a relatively small set of genes or targeted
regions of interest. The fast turnaround time, low cost, low
sample input requirement, and relative ease of interpretation
make targeted sequencing particularly well suited for both
translational and clinical research applications. Sanger
sequencing is often used to confirm variants identified by NGS.
Cloning

• Gene cloning, which creates copies of genes or segments of

DNA.
• Reproductive cloning, which creates copies of whole animals.
• Therapeutic cloning, which creates embryonic stem cells.
Gene cloning
• Restriction enzme based-gene cloning
• TA/TOPO cloning
• Gateway cloning
• Infusion cloning
• Ligation-independent cloning
• Bi/multi-cistronic cloning
• Gibson assembly
• Golden gate cloning

https://info.gbiosciences.com/blog/8-techniques-to-clone-a-
gene-which-method-is-the-best-for-you
Restriction Cloning
TOPO/TA-cloning
Gateway Cloning
Gateway BP Clonase catalyzes the
in vitro recombination of PCR
products or subcloning DNA
segments from clones (containing
attB sites) and a donor vector
(containing attP sites) to generate
entry clones with attL sites

Gateway LR Clonase catalyzes the

in vitro recombination between an
entry clone (containing a gene of
interest flanked by attL sites) and a
destination vector (containing attR
sites) to generate an expression
clone.
Infusion Cloning
Ligation-independent cloning

T4 polymerase has 3’ to
5’ exonuclease activity
resulting in overhangs
that are complementary
to both gene and vector.
Bi/Multi-cistronic cloning
Internal ribosome entry site (IRES) elements
- In eukaryotes translation occurs only from 5’end
hence only one translation end. But this IRES
elements have the capability to start translation
irrespective of 5’end. So, if this IRES elements are
incorporated between two genes both can easily
be expressed. But this method has few
disadvantages like their large size (500bp), often
the second gene express less compared to the
first

2A peptides - Small peptides of 20 aa length are

used by researchers to overcome some of the
problems faced by using IRES. 2A peptide
sequences usually start with GSG residues and
end with PGP, where the cleavage of the second
protein occurs between G and P and the second
one will start with proline residue. This
technique is successfully used to clone more
than two genes in a single multi-cistronic
Gibson Assembly
Golden gate cloning
Two enzymes are used in this cloning method, Type II restriction enzymes and
DNA ligase enzyme. Type II restriction enzymes like Bsa I, BsmB I, Bbs I. These
enzymes will cut the DNA fragment outside the recognition sequence resulting in
non palindromic over hangs.

This is an irreversible method of gene assembly, once the gene is inserted in the
vector it can never be cut with the same restriction enzyme