Notesale: Turn your study into money

Document Preview

Extracts from the notes are below, to see the PDF you'll receive please use the links above

---

Genetic
Engineering
Book list:
1
...
C Dubey

2
...
Satyanarayana

3
...
i Genetics
By Russel

1

1
...
It refers to the different techniques used to isolate, manipulate, amplify, analyze, recombine and
propagate a specific DNA segment
...

2
...


4
...

6
...


Isolation of a target gene: The target gene is cleaved by restriction endonuclease enzymes that cut DNA
chains at specific locations and produce sticky ends
...
The vector DNA is also
cut by restriction endonuclease enzymes producing sticky ends that are complementary to the sticky ends
of target DNA
...
g
...
g
...
The hybrid combination of two fragments of DNA
is called recombinant DNA or chimeric DNA (hybrid plasmid)
...
g
...
coli) by
transformation or by viral infection
...

Bacteria are lysed by using appropriate antibiotics and the hybrid plasmids are isolated
...

Isolation of target DNA by southern blot technique and identification of target DNA by DNA probe
...


Biomedical importance: Recombinant DNA technology has a wide range of applications, such as –
1
...

3
...

5
...

7
...


It offers a rational approach to understanding the molecular basis of a number of diseases e
...
sickle cell
anemia, thalassemia, familial hypercholesterolemia, cystic fibrosis, muscular dystrophy
...
g
...

Production of proteins for vaccines e
...
hepatitis B vaccine and for diagnostic testing e
...
AIDS tests
...

Application of forensic medicine, e
...
identification of criminals, paternity tests
...

Prenatal diagnosis of genetic diseases
...


Recombinant DNA or chimeric DNA: Recombinant DNA is the DNA constructed by combination of different
DNA fragments of different sources
...

Restriction site: A specific DNA sequence that is recognized by a restriction enzyme called a restriction site
...
e
...

Restriction fragment: Restriction enzyme cleaves DNA at a specific nucleotide sequence and produce precisely
defined DNA segments called restriction fragments
...

Sticky ends: Some restriction endonucleases form staggered cuts that produce sticky or cohesive ends that is, the
resulting restriction fragments have single stranded sequences that are complementary to each other
...

Blunt ends: Some other restriction endonucleases cleave in the middle of their recognition sequence and produce
restriction fragments that have blunt ends that do not form hydrogen bonds with each other
...
It is also called restriction enzyme
...

2
...


Restriction endonucleases are produced by bacteria
...

These enzymes were called restriction enzymes because their presence in a given bacterium restricted the
growth of certain bacteriophage viruses by cleaving their DNA
...
This includes the coding
region itself as well as associated regulatory regions
...

Heredity: Heredity can be defined as a process of passing biological traits or characteristic from parents to their
offspring
...
It
seeks to understand the process of trait inheritance from parents to offspring, including the molecular structure and
function of genes, gene behavior in the context of a cell or organism (e
...
dominance and epigenetics), gene
distribution, variation and change in populations
...

Properties: Essential properties of a vector include:
1
...

3
...


Capable of autonomous replication
...

Must contain at least one specific nucleotide sequence recognized by a restriction endonuclease
...


Commonly used vectors:
1
...

3
...


Plasmid: Plasmids accept DNA fragments about 6-10 kb long
...
Phases can accept DNA fragments 10-20 kb long
...

Mammalian viruses e
...
retroviruses
...
Growing and harvesting a bacterial culture: Most bacteria can be grown without too much difficulty in
a liquid medium (broth)
...

Luria Bertani (LB) is a complex or undefined medium, meaning that the precise identity and quantity of
its components are not known
...
Preparation of a cell extract: Techniques for breaking open bacterial cells can be divided into –
Physical method: In this method, the cells are disrupted by mechanical forces
...
Chemical methods are most commonly used with bacterial cells when the
object is DNA preparation
...
Lysis: Lysis refers to the breaking of the cell wall and cellular membranes (especially, the plasma and
nuclear membranes)
...

Chemical lysis generally involves one agent attacking the cell wall and another disrupting the cell
membrane
...

For cell membrane lysis, sodium dodecyl sulphate (SDS) is used
...
Purification of DNA from cell extract: In addition to DNA, a bacterial cell extract contains significant
quantities of protein and RNA
...

Ion exchange chromatography to separate the mixture into its various components
...

↓

3

5
...
Absolute
ethanol is layered on top of a concentrated solution of DNA
...
For less concentrated solutions, ethanol is added at a ratio of 2
...
Precipitated DNA is collected by centrifugation
...
The amount of UV radiation absorbed by a solution of DNA is directly proportional
to the amount of DNA in the sample
...
Instead, a different
approach must be used
...

When CTAB is added to a plant cell extract, the nucleic acid-CTAB complex precipitates, leaving carbohydrate,
protein and other contaminants in the supernatant
...

↓
The nucleic acids can now be concentrated by ethanol precipitation and the RNA removed by ribonuclease
treatment
...
In a plasmid preparation, it is always necessary to separate the plasmid DNA from the large amount
of bacterial chromosomal DNA that is also present in the cells
...

•
•

Plasmids and bacterial DNA differ in size
...


A
...
coli chromosome and most are much
smaller than this
...

Procedure:
If the cells are lysed under very carefully controlled conditions, only a minimal amount of chromosomal
DNA breakage occurs
...

↓
Cell disruption is carried out very gently to prevent wholesale breakage of the bacterial DNA
...

↓
Spheroplasts are formed (cells with partially degraded cell walls that retain an intact cytoplasmic
membrane)
↓
Cell lysis is now induced by adding a non-ionic detergent such as Triton X – 100 (ionic detergents such
as SDS causes chromosomal breakage)
...

↓
If the plasmids themselves are large molecules, they may also sediment with the cell debris
...
Separation on the basis of conformation: The term conformation refers to the overall spatial configuration
of the molecules, with the two simplest conformations being linear and circular
...
Molecule supercoiling occurs because the double helix of
the plasmid DNA is partially unwound during the plasmid replication process by enzymes called

4

topoisomerases
...
If one of the polynucleotide
strands is broken, the double helix reverts to its normal relaxed state, and the plasmid takes on the
alternative conformation, called open circular
...
It can be done by –
1
...
Ethidium bromide – caesium chloride density gradient centrifugation
...
Alkaline denaturation:
The basic of the technique is the use of narrow pH range at which non-supercoiled DNA is denatured,
whereas supercoiled plasmids are not
...
5
...

↓
Acid is now added so that the denatured bacterial DNA strands re-aggregate into a tangled mass
...


2
...
This process is referred as isopycnic centrifugation
...
e
...
7 g/cm3 in the
gradient
...

↓
EtBr binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding
of the double helix
...
125 g/cm 3 for linear
DNA
...

↓
The decrease in buoyant density of a supercoiled DNA is much less only about 0
...
As a
consequence, supercoiled DNA form a band in an EtBr-CsCl2 gradient at a different position to linear
and open-circular DNA
...

↓
The position of the DNA bands can be seen by shining UV radiation on the tube, which causes the
bound EtBr to fluoresce
...

↓
The EtBr bound to the plasmid DNA is extracted with n-butanol and the CsCl2 removed by dialysis
...

Density gradient centrifugation in the presence of ethidium bromide (EtBr) can be used to separate
supercoiled DNA from non-supercoiled DNA
...
Some multi copy plasmids
(those with copy numbers of 20 or more) have the useful property of being able to replicate in the absence of protein
synthesis
...
g
...

↓
During this time, the plasmid molecules continue to replicate, even though the chromosome replication and
cell division are blocked
...
Amplification is therefore, a
very efficient way of increasing the yield of multi copy plasmids
...
This is because bacteriophage
particles can be obtained in large numbers from the extracellular medium of an infected bacterial culture
...
The phage particles
are then collected from the suspension and their DNA extracted by a single deproteinization step to remove the
phage capsid
...

↓
The remains of lysed bacterial cells, along with any infected cells are removed from an infected culture by
centrifugation, leaving the phage particles in suspension
...
Collection of phages
is therefore, usually achieved by precipitation with polyethylene glycol (PEG)
...

↓
The precipitate can then be collected by centrifugation, and re-dissolved in a suitably small volume
...
This is necessary because the PEG
precipitate also contains a certain amount of bacterial debris, possibly including unwanted cellular DNA
...
45 – 1
...

↓
Removal of CsCl2 by dialysis leaves a pure phage preparation from which the DNA can be extracted by
either phenol or protease treatment to digest the phage protein coat
...
Describe briefly the purification of M13 DNA (M13 is a non-lysogenic λ phage virus)
...
Then centrifugation is done
...


7

2
...

Types: 2 types:
1
...


Genomic DNA library and
Complementary DNA library

They are discussed below:
Genomic DNA library: It ideally contains a copy of every DNA nucleotide sequence in the genome
...

2
...


It is the collection of cloned restriction fragments of the entire DNA of an organism
...

Building of genomic library involves four steps:
i
...

ii
...

iii
...

iv
...


Complementary DNA (cDNA) libraries: cDNA libraries contain those DNA sequences that appear as mRNA
molecules
...

2
...


They differ from one cell type to another
...

It is more specialized and expensive
...
In recombinant DNA technology, scientists use gel electrophoresis to isolate fragments of
DNA molecules that can then be inserted into vectors, multiplied by PCR or preserved in a gene library
...
The gel is typically
composed of agar, called agarose, which acts as a molecular sieve that retards the movement of DNA fragments
down the chamber and separates the fragments by size
...
Scientists can determine the size of a fragment by comparing the distance it travels to the distances travelled
by standard DNA fragments of known sizes
...
Scientists could also use
probes to localize specific sequences in electrophoresis gels, but because gels are flimsy and easily broken and
deform as they dry, it is difficult to probe gels
...


Blotting techniques
Blotting techniques are very widely used analytical tools for the specific identification of desired DNA or RNA
fragments from thousands of molecules
...
The blotted nucleic acids are then used as targets in the
hybridization experiments for their specific detection
...

2
...

4
...

6
...


Southern blotting (for DNA)
Northern blotting (for RNA)
Dot blotting (DNA/RNA)
Western blotting (for protein)
Colony and plaque hybridization (cell from colony)
Eastern blotting
Far eastern blotting

They are discussed below:
General steps:

Southern blotting:
Steps:
DNA molecule
↓
Restriction enzymes
Restriction fragments
↓
Gel electrophoresis is used to separate fragments by size
...

The DNA fragments are invisible to the investigators at this stage
↓
Nitrocellulose membrane with DNA fragments at same locations as in gel (still invisible) is baked to
permanently affix DNA
...

↓
Film is developed
...


Stringent conditions (stringency control): Stringency refers to the specificity with which a particular DNA
target sequence is detected by a probe
...


salt concentration) only completely complementary DNA sequences will bind and hybridize
...
Therefore, stringency
control is very essential for specific detection of DNA molecules in Southern blotting
...

Membranes for blot transfer: In the early years, nitrocellulose was used for immobilization of DNA
molecules during blot transfer
...
In recent years, most laboratories use nylon membranes as they possess high tensile strength and
better binding capacity for nucleic acids
...

2
...

4
...

6
...

It is important for the confirmation of DNA cloning results
...

Forensically applied to detect minute quantities of DNA to identify parenthood, thieves, rapists etc
...

DNA pieces from one species (e
...
human) can be used to detect DNA molecules from related species (e
...

chimpanzee, cow)
...


Northern blotting: Northern blotting is the technique for the specific identification of RNA molecules
...

Steps:
RNA isolation
↓
Loading of sample on Agarose gel
↓
Blotting on nitrocellulose membrane
↓
Labeling with probe
↓
Washing to remove unbound probe
↓
Detection by autoradiogram
...

2
...

4
...

6
...

Detection of mRNA transcript size
...

Study RNA splicing – can detect alternatively spliced transcripts
...

Study IRES (Internal Ribosomal Entry Site) to remove possibility of RNA digestion vs 2 nd cistron
translation
...

2
...

4
...

RNA samples can be degraded by RNAses
...

Detection with multiple probes is a problem
...
In this approach, the
nucleic acids (DNA or RNA) are directly spotted onto the filters and not subjected to electrophoresis
...

Dot blotting technique is particularly useful in obtaining quantitative data for the evaluation of gene expression
...

Principle of western blotting: Western blotting (protein blotting or immunoblotting) is a rapid and sensitive assay
for detection and characterization of proteins
...

The proteins thus separated are then transferred or electrotransferred onto nitrocellulose membrane and are
detected using specific primary antibody and secondary enzyme labeled antibody and substrate
...

↓
Transfer of electrophoresed gel in a buffer at low temperature (400 C) for half an hour
↓
Blotting of proteins onto nitrocellulose filter paper
...

↓
Putting the complete set up in transfer tank containing sufficient transfer buffer
↓
Application of an electric field (30 V overnight for 5 hours) to cause migration of proteins from the gel to
nitrocellulose filter and binding on its surface (the nitrocellulose filter has exact image of pattern of proteins as
present in the gel
...

↓
Hybridization of proteins by using radiolabeled antibodies (I123 – antibodies) of known structure, isolated from
the rabbit
...

↓
Detection of hybrid sequences by autoradiography
...

[From: Textbook of Biotechnology by R
...

Dubey
...

2
...

4
...


Confirmatory of HIV test
...

Some forms of Lyme disease testing employs western blotting
...

In biochemistry, it is used to study different properties of protein based on molecular weight
...

Process:

Eastern blotting:
The eastern blotting is a biochemical technique used to analyze protein post translational modifications (PTM)
such as lipids, phosphomoieties and glycoconjugates
...
Thus,
eastern blotting can be considered an extension of the biochemical technique of western blotting
...

Applications:
1
...

3
...
g
...
muris and Ixodes ovatus ticks (IOE)
...
muris is more post translationally
modified than the highly virulent IOE
...


Far eastern blotting: Far eastern blotting is a technique developed in 1994 by Taki and colleagues at the Tokyo
Medical and Dental University, Japan for the analysis of lipids separated by high – performance thin layer
chromatography (HPTLC)
...
g
...
It is based on the
technique of western blotting to detect protein-protein interaction in vitro
...

Thus, whereas western blotting is used for the detection of certain proteins, far western blotting is rather employed
to detect protein – protein interactions
...
PCR is based on using the
ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand
...
DNA polymerase then elongate its 3 end by adding more nucleotides to generate an extended region
of double stranded DNA
...

2
...

4
...

6
...

8
...

10
...

12
...

14
...

16
...

18
...

20
...

22
...


Real-time PCR
Quantitative real time PCR (Q-RT PCR)
Reverse Transcriptase PCR (RT-PCR)
Multiplex PCR
Nested PCR
Long-range PCR
Single-cell PCR
Fast-cycling PCR
Methylation-specific PCR (MSP)
Hot start PCR
High-fidelity PCR
In situ PCR
Variable Number of Tandem Repeats (VNTR) PCR
Asymmetric PCR
Repetitive sequence-based PCR
Overlap extension PCR
Assemble PCR
Intersequence-specific PCR(ISSR)
Ligation-mediated PCR
Methylation –specifin PCR
Miniprimer PCR
Solid phase PCR
Touch down PCR, etc

Requirement:
1
...

3
...


Target DNA
Taq DNA polymerase
A pair of primers
All four deoxyribonucleotides (dNTP): dATP, dTTP, dGTP, dCTP
...

2
...

4
...

Priming: A mixture containing an excess of DNA primers (synthesized such that they are complementary
to nucleotide sequences near the ends of the target DNA), DNA polymerase and an abundance of the four
deoxyribonucletide triphosphates (A, T, C and G) is added to the target DNA
...
Because there is an excess of primers, single
strands are more likely to bind to a primer than to one another
...

Extension: Raising the temperature to about 720 C increases the rate at which DNA polymerase replicates
each strand to produce more DNA
...
After
only 30 cycles – which requires only a few hours to complete – PCR produces over 1 billion identical copies
of the original DNA molecule
...
These include:
1
...


2
...

4
...

6
...

8
...


Identification of criminals by amplifying DNA of forensic samples (e
...
human hair, blood or
semen) collected from crime scenes
...

Verification of paternity
...
This helps early and confirmatory diagnosis of infectious
diseases like AIDS, TB etc
...

Prenatal diagnosis of genetic diseases e
...
cystic fibrosis
...

Cancer detection by detecting mutation in oncogene or tumor suppressor gene
...
But it is costly, so we use HLA typing for this purpose
...

DNA analysis
...

2
...

4
...
Even a trace amount of target DNA can be amplified
...

Simplicity: It is technically simple and less difficult than biological cloning of DNA
...


Disadvantages of PCR:
1
...

3
...


Base sequence of the flanking region must be known to construct primer
...

Inaccuracy: Taq DNA polymerase has no proof reading activity
...

False positivity: Because of high sensitivity, very small contamination of DNA from other sample can
give false positive result
...


It is natural, in vivo process
...


It is artificial, in vitro process
...


It is cell based process
...


It is enzyme based, cell free process
...


Needs host cell and vector
...


Does not need host cell and vector
...


Taq DNA polymerase is not needed
...


Taq DNA polymerase is needed
...


It is slow, complex and expensive
...


It is rapid, simple and less expensive
...


There is no need to know the flanking
sequence of target DNA
...


There is need to know the flanking sequence
and target DNA
...


It is less sensitive
...


It is more sensitive
...


Large target DNA can be cloned
...


Only small target DNA can be cloned
...
It is also called site-specific
mutagenesis or oligonucleotide-directed mutagenesis; is used for investigating the structure and biological activity
of DNA, RNA, and protein molecules, and for protein engineering
...
It is complementary to a position of a gene around the site to be mutated
...

↓
The starting material is a single-stranded DNA (to be mutated) carried in an M13 phage vector
...

↓
Hybridization (despite a single base mismatch) is possible by mixing at low temperature with excess of primer, and
in the presence of high salt concentration
...

↓
The oligonucleotide primer is extended to form a complementary strand of the DNA
...

↓
The double stranded DNA (i
...
M13 phage molecule) containing the mismatched is introduced into E
...

↓
The infected E
...

↓
The virus particles lyse the cells and form plaques
...

↓
The double stranded DNAs of M13 are isolated
...
coli
...
coli which can be isolated and purified
...

2
...


Multiple point mutagenesis: Oligonucleotide directed mutagenesis can be used to create DNAs with
multiple point mutations with the requisite number of base mismatches
...
This can bind with target DNA on
either side
...
This enables a small position of target DNA to be deleted
...
DNA fingerprinting is the present day genetic detective in the practice of modern medical
forensics
...

↓
The DNA is transferred to nylon
...

↓
Adding radioactive or colored probes to the nylon sheet produces a pattern called the DNA fingerprint
...

Applications:
1
...

3
...

Reuniting the lost children: DNA profiling technology helpes in reuniting the lost children with their
respective parents or vice versa
...


16

4
...

6
...

Food industry: In food industry, DNA markers are used for authentication of food products
...
This helps in improving the quality of agricultural products
...
The
following there methods are used for the determination of DNA sequences
...
Maxam and Gilbert’s chemical degradation method:
Steps:
The 3/ ends of DNA are labeled with 32P
...

↓
The mixture is divided into four samples, each treated with a different reagent having the property of
destroying either only G, or only C, or A and G or T and C
...

↓
Each of the four samples is electrophoresed in four different lanes of the gel
...

2
...

↓
A small amount of ddATP is added to tube 1, ddGTP to tube 2, ddTTP to tube 3 and ddCTP to tube 4
...

↓
DNA polymerase is added to each tube, DNA synthesis starts and chain elongates
...
The length of the fragment
depends on the position of incorporation of ddNTPs
...

↓
Then the gel is dried and autoradiography is done so that position of different bands (having radiolabeled
5/ end) in each lane is observed
...

↓
DNA sequence is obtained by reading (from bottom to top of the gel) the bands on autoradiogram of four
lanes
...

Direct DNA sequencing using PCR: From above
...
Mapping Genomes and Protocols of
Detection of Polymorphisms
Gene mapping: Gene mapping can be defined as assigning or locating a specific gene to particular region of a
chromosome and determining the location of and relative distances between genes and chromosomes
...


2
...

4
...

6
...


Genome mapping provides the basis for whole genome sequencing and the Human Genome Project
...

It is essential for human genetics for the identification of genes associated with heredity diseases
...

To assign the genes to a particular chromosome
...

To learn about other important parts of the genome, such as the regulatory regions that help control when
genes are truned on and off
...


Types of gene mapping:
1
...


Genetic mapping and
Physical mapping

They are discussed below:
1
...
A genetic
map is also called a linkage map or a chromosome map
...
Genetic
techniques include cross-breeding experiments or in the case of humans, the examination of family
histories
...

↓
These are referred to as markers and are extremely valuable for tracking inheritance of characteristics or
diseases through several generations of a family
...

↓
If a particular gene is close to a DNA marker on the chromosome, it is more likely that the gene and marker
will stay together during the recombination process and are therefore, more likely to be passed down along
the family line (inherited) together
...
The more DNA markers there are

18

on a genetic map, the more likely it is that one of them will be located close to the disease or trait associated
gene
...

ii
...

iv
...


2
...

The resolution of a genetic map depends on the number of crossovers that have been scored
...

Have limited accuracy
...


Physical mapping: Physical mapping gives an estimation of the (physical) distance between specific
known DNA sequences on a chromosome
...

Classification or types:
•
•
•

Chromosomal or cytogenic maps
Radiation hybrid (RH) maps and
Sequence maps

Techniques: There are several different techniques used for physical mapping
...

Similarities between genetic map and physical map:
1
...


Differences between genetic map and physical map: DAREM
Characteristics
1
...
Accuracy
3
...
Efficiency

Genetic map
A gene map based on the gene linkage
and gene association studies done on a
genetic marker or gene loci of a
chromosome
...

Less efficient

19

Physical map
A gene map where gene map is physically
derived by isolating the DNA and obtaining
precise genetic marker using molecular
biology techniques
...

Highly efficient

5
...


Mendelian

inheritance

Not directly based on Mendelian genetics
...

Principles:
Basic elements: DNA probe and a target sequence
...

↓
The DNA probe is labeled in various ways such as nick translation, random primed labeling and PCR
...

• In case of a direct labeling, probes are being labeled with nucleotides containing a fluorophore
...

↓
The labeled probes and the target DNA are denatured
...

↓
In the case of indirect annealing, an extra step in needed for visualization of non-fluorescent hapten that uses an
enzymatic or immunological detection system
...

Applications:

1
...
Gene mapping
3
...

4
...

5
...

6
...

7
...

8
...

9
...

10
...

Advantages:

1
...

3
...
This is useful for the visualization of chromosomal aberrations directly in cytological
preparations and tissue sections
...

Material for FISH can be processed in 4-24 hours and the analysis of 1000 – 2000 cells accomplished in
15-45 min, enabling the information on the cytogenetic pattern of tumor cells to be achieved within a
sufficient time frame for use in treatment strategies
...


20

Applications of RFLP:

1
...

3
...


To determine the status of genetic diseases such as Cystic fibrosis in an individual
...

In genetic mapping: To determine recombination rates that show the genetic distance between the loci
...


Limitations:

1
...

2
...

3
...

Due to such problems, the PCR technique has largely replaced RFLP in most applications requiring DNA
sequencing such as paternity testing or forensic sample analysis
...
Cloning Vectors
Vector: A vector is a DNA molecule which is used for transporting exogenous DNA into the host cell
...

Cloning vector: A cloning vector is a fragment of DNA which is capable of self-replication and stable maintenance
inside the host organism
...
Most of the cloning
vectors are genetically engineered
...

Distinct features of cloning vectors:

1
...

3
...


5
...

7
...
The foreign DNA starts replicating along with the host cell when it is integrated
or connected to this sequence
...
g
...

Presence of restriction sites: It should have restriction sites to enable break up of certain sequences with
respect to restriction endonuclease
...
By inactivating
it, through restriction enzyme one can detect the recombinant molecules
...

These are called multiple cloning sites (MCS) or polylinkers
...

Multiple cloning sites: There must be multiple sites for cloning
...
so that the cloned DNA should express properly
...


Types of cloning vectors:
A
...
Plasmids e
...
pBR 322, pUC 18
2
...
Bacteriophage lambda
4
...
Phagemids
6
...
Eukaryotic cloning vectors:
1
...
Yeast artificial chromosome (YAC)
3
...
Prokaryotic cloning vectors:
Plasmids:
Characteristics:

1
...

2
...
However, not all plasmids are circular in origin
...
They are present in bacteria, archaea and eukaryotes
...
The size of plasmids ranges from 1 kb to 250 kb
...
DNA insert of up to 10 kb can be cloned in the plasmids
...
The plasmids have high copy number which is useful for production of greater yield of
7
...


recombinant plasmid for subsequent experiments
...

Plasmids only encode those proteins which are essential for their own replication
...


Examples: pBR322, pUC18, F plasmid, Col plasmid
...

Advantages of using plasmids as vectors:

1
...

3
...

5
...

More stable because of circular configuration
...

High copy number
...


Disadvantages of using plasmids as vectors:

1
...

2
...

3
...

Bacteriophage
Characteristics:

1
...

3
...


Bacteriophages or phages are viruses which infect bacterial cells
...

A maximum of 53 kb DNA can be packaged into the phage
...


Examples: Phage λ, M13 coliphage etc
...
They are more efficient than plasmids for cloning large inserts
...
Screening of phage plaques is much easier than identification of recombinant bacterial colonies
...

2
...

4
...

Phagemid contains the F1 origin of replication from F1 phage
...

It replicates as a plasmid and gets packaged in the form of single stranded DNA in viral particles
...
They contain multiple cloning sites
...
An inducible Lac gene promoter is present
...
Blue white colony selection is observed
...
Cosmids are plasmids
...
They are capable of incorporating the bacteriophage λ DNA segment
...


3
...

4
...

5
...
This permits the foreign DNA fragment or genes to be introduced
into the host organism by the mechanism of transduction
...
They have high transmission efficiency and are capable of producing a large number of clones
from a small quantity of DNA
...
Also they can carry up to 45 kb of insert compared to 25 kb carried by plasmids and λ
...
Cosmids cannot accept more than 50 kb of the insert
...
Retroviruses are the virus with RNA as the genetic material
...
Retroviral vectors are used for introduction of novel or manipulated genes into the animal or
human cells
...
The viral RNA is converted into DNA with the help of reverse transcriptase and henceforth,
efficiently integrated into the host cell
...
Any gene of interest can be introduced into the retroviral genome
...

Advantages:

1
...

The things which matter while choosing cloning vector are:

1
...

3
...


DNA inset size
...

Restriction size
...


B
...
Bacterial artificial chromosomes are similar to E
...

24

2
...
These proteins are critical for BAC
replication
...
It is derived from naturally occurring F plasmid
...
The DNA inset size varies between 150 to 350 kb
...
They are capable of accommodating large sequences without any risk of rearrangement
...
BACs are frequently used for studies of genetic or infectious disorders
...
High yield of DNA clones is obtained
...
They are present in low copy number
...
The eukaryotic DNA inserts with repetitive sequences are structurally unstable in BACs often
resulting in deletion or rearrangement
...
A large DNA inset of up to 200 kb can be cloned
...
They are used for cloning inside eukaryotic cells
...

4
...

6
...

It possesses the yeast telomere at each end
...

The Ori is bacterial in origin
...


Advantages:

1
...

2
...

Disadvantages:

1
...

2
...

Advantages of BACs over YACs:

1
...

3
...

5
...

Easy to transform
...

User friendly
...


Human artificial chromosome (HAC)
Characteristics:

1
...

3
...


Human artificial chromosomes are artificially synthesized
...

It can carry large amounts of DNA inserts
...


Advantages:

25

1
...

2
...


26

5
...
It is the process of developing useful or valuable proteins
...
e
...

↓
Synthesis of desired DNA or site-directed mutagenesis of desired gene to get expected new engineered protein
...

Objectives of protein and enzyme engineering:
1
...

3
...

To produce enzymes and proteins in large quantities
...


Properties required for protein (enzyme) engineering:
Optimum pH
Thermos stability
Substrate specificity
Co-factor requirements
Protease resistance
Kinetic properties and Michaels constant (Km)
Allosteric regulation
Molecular weight and structure of subunits

1
...

3
...

5
...

7
...


Applications of protein engineering:

1
...

Proteases are used in several applications of food industry regarding low allergenic infant formulas,
milk clotting and flavors
...

Amylases are used for liquefaction and saccharification of starch as well as in adjustment of flour and
bread softness and volume in baking
...

Lipases are used in many applications of food industry such as for the stability and conditioning of
dough and in cheese flavor applications
...
Detergent industry:
i
...

ii
...

3
...


27

i
...


5
...


7
...

ii
...

Medical applications:
i
...

ii
...

iii
...

iv
...

v
...

vi
...

vii
...

Biopolymer production:
i
...
Particularly,
pesticides are becoming increasingly important as biomaterials because of their specific physical,
chemical and biological properties
...

Protein engineering and macromolecular self-assembly are utilized to produce peptide based
materials, such as elastin like polypeptides, silk like polymers
...

The ability of protein engineering to create and improve protein domains can be utilized for
producing new biomaterials for medical and engineering applications
...

Nano biotechnology for protein engineering are becoming increasingly important
...

ii
...

iii
...
Thus, they are
suitable components for controlled synthesis and assembly of nanotechnological systems
...

Combinational biology methods commonly applied in protein engineering studies, such as phage
display and bacterial cell surface display technologies, are also used to select polypeptide
sequences which selectively bind to inorganic compound surfaces, for ultimate applications of
nanobiotechnology
...

Another increasing nanobiotechnology application is the use of amyloid fibrils as structural
templates for nanowire construction
...

vi
...

Other applications:
i
...

ii
...
The zinc finger design and principle is used to design DNA binding proteins to
control gene expression
...

Virus engineering is another emerging field, where the virus particles are modified by protein
engineering
...
They could be used as new vaccines, gene therapy and targeted drug delivery
vectors, molecules imaging agents and as building blocks for electronic nanodevices or
nanomaterials construction
...

Protein cysteine modifications are also important protein engineering applications
...
This method could be potentially used for development

28

of new protein-based drugs, improving their half-life, reducing their toxicity and preventing
multidrug resistance development
...
Cell and Tissue Culture
Cell culture: Cell culture is the process by which prokaryotic, eukaryotic or plant cells are grown under controlled
conditions
...
Cell culture was first
successfully undertaken by Ross Harrison in 1907
...

Culture media: Choice of media depends on the type of cell being cultured
...

Prepared medium is filtered and incubated at 40 C
...
Cell to be kept in healthy and growing state have to be sub-cultured or passaged
...
Enzymes such as trypsin, dipase, collagenease in
combination with EDTA breaks the cellular glue that attached the cells to the surface
...
Human cell lines

2
...


• MCF 7: Breast cancer
• HL 60: Leukemia
• HEK 293: Human embryonic kidney
• HeLa: Henrietta lacks
Primate cell lines:
• Vero: African green monkey kidney epithelial cells
...

Insect cell lines:
• Sf 9 and
• Sf 21

Contaminants: Cell culture contaminants are of two types:

1
...
Chemical contamination is caused by endotoxins,
2
...

Biological contaminants: They cause visible effects on the culture
...


Effects of biological contaminants:

1
...

3
...


They compete for nutrients with host cells
...

They degrade arginine and purine that inhibit the synthesis of histone and nucleic acid
...


Detection of contaminants:

1
...

3
...


• Inclusion bodies and
• Cell lysis
Yeasts, bacteria and fungi usually shows visible effects on the culture (changes in medium turbidity or pH)
...
g
...

Mycoplasma can also be detected by enzyme immunoassay by specific antisera or monoclonal antibodies
or by PCR amplification of mycoplasmal RNA
...

Minimal requirements for the cell culture:
Infrastructure:
1
...

3
...

5
...


Equipment:
1
...

3
...

5
...

7
...

9
...

11
...

13
...

15
...

17
...

19
...

21
...


Laminar flow
Sterilizer
Incubator
Refrigerator and freezer (-200 C)
Balance
CO2
Cylinder
Centrifuge
Inverted microscope
Water purifier
Hemocytometer
Liquid nitrogen freezer
Slow cooling device for freezing cells
Pipette washer
Deep washing sink
Air conditioned rooms
Contaminant room for biohazard work
Phase contrast microscope
Confocal microscope
Osmometer
High capacity centrifuge and
Time lapse video equipment

Rules for working with cell culture:

1
...

2
...

•
•
•
•
•
•

Diploid cells (primary cultures, lines for the production of vaccines etc
...

Lines which may be contaminated
Virus producing lines
...
If working on the bench, Bunsen flame should be used to heat the air surrounding the Bunsen
...
All bottle tops and necks should be swabbed with 70% ethanol
...
All bottle necks and pipettes should be flamed by passing very quickly through the hottest part of the flame
...
Placing caps and pipettes down on the bench should be avoided
...

6
...

One should work either left to right or vice versa, so that all material goes to one side, once finished
...


Safety aspect in cell culture:

1
...

2
...
If caps are dropped or bottles are
unconditionally touched, they should be replaced with new ones
...
Necks of glass bottles should be heated at least for 60 seconds at a temperature of 200 0 C
...
The laminar flow should be switched on 20 minutes prior to start working
...
Cell cultures which are frequently used should be subcultured and stored as duplicate strains
...
Actively growing cells that are in their log phase of growth, should be used
...
Exposure to trypsin should be kept at minimum
...
The cells should be handled gently
...

9
...

10
...
For
slowly growing cells, 105 cells/ml should be used
...
A callus is produced
when explants (cells) are cultured in an appropriate medium
...

Stem Cell Culture:
Human embryonic stem cells (hESCs) are generated by transferring cells from a pre-implantation stage embryo into
a plastic laboratory culture dish that contains a nutrient broth known as culture medium
...

↓
However, if the plated cells survive, divide and multiply enough to crowd the dish, they are removed gently and
plated into several fresh culture dishes
...
Each cycle of
sub-culturing the cells is referred to as a passage
...

↓
Embryonic stem cells have proliferated in cell culture for six or more months without differentiating, are
pluripotent, and appear genetically normal are referred to as an embryonic stem cell line
...

Plant cell and tissue culture:
Major steps of tissue culture:
Stage 1: Initiation phase: The initiation phage is the first phase of tissue culture
...

In this stage, the tissue is initiated in to culture
...
Here, the medium is composed of appropriate components for growth including regulators and nutrients
...
This step is often
repeated several times in order to obtain the desired number of plants
...
Here, hormones are required in order to induce
rooting and consequently complete plantlets
...
Here, NaOH or HCl is used to
adjust the pH
...

↓
After cooling, the warm medium is poured into polycarbonate tubes to a depth of about 4 cm
...

↓
The plant is cut into small pieces
...

↓
The plant part is transferred into sterilizing Clorox solution, shook for 1 minute and leaved to stock for 20 minutes
...

↓
The plant material is transferred to a tissue culture medium
...

↓
The container is opened and sterile water is poured to cover half of the container
...

↓
The water is poured and this is repeated for three times
...

↓
Using a sterile blade, the plant material is cut into smaller pieces of about 2-3 mm across avoiding the parts that
have been damaged by bleach
...
Depending on the plant used, it is important
to check and find out how it should be placed in the medium
...
Once the shoots develop, then the plant section may be placed in the right
environment for further growth
...

Techniques for in vitro plant culture:

1
...
This
2
...


has the potential to provide rapid and large scale propagation of new genotypes
...

Transgenic plants: It is used for expression of mammalian genes or plant genes for various species
...


33

7
...

2
...

4
...

6
...

8
...

10
...

Acquire insecticidal resistance
...

Develop the plant to grow in draught
...

Make the plants to grow in various seasons
...

Resistance against fungal pathogens
...

Delayed ripening can be done which is beneficial in many respects
...
Physical methods: These include –
1
...
Electroporation
3
...
Pressure
5
...
Laser mediated
B
...
PEG (polyethylene glycol)
2
...
DAE-dextrin
4
...
Proteins
6
...
Biological methods:
1
...
Virus mediated
They are discussed below:
A
...
Microinjection: Microinjection is the method tried for artificial DNA transfer to cereal plants
that show inability to regenerate and develop into whole plants from cultured cells
...
3 ml of DNA solution is injected at a point above tiller node until several
drops of solution came out from top of young inflorescence
...

Advantages:
1
...

2
...

3
...

4
...

5
...

6
...

7
...

Disadvantages:
1
...


34

2
...

4
...

Frequency of transformation is very low
...


2
...

Process:
Cultured cells
↓
Mix cells and DNA in a special cuvette
↓
Application of voltage
↓
Cells take up DNA through holes in membrane
↓
DNA enters nucleus
↓
Gene expression

3
...
It is the simplest method of direct introduction to therapeutic DNA into target cells
...

Process:
The gene gun is a device that literally fires DNA into target cells
...
Beads are carefully coated with DNA
...

↓
An explosive force fires the bullet down the barrel of the gun towards the target cells that lie just
beyond the end of the barrel
...
Some of the beads pass through the cell wall into the cytoplasm
of the target cells
...

↓
Once the inside the target cells, the DNA is solubilized and may be expressed
...
Requirement of protoplast can be avoided
...
Walled intact cells can be penetrated
...
Manipulation of genome of subcellular organelles can be achieved
...

2
...

4
...


Shallow penetration of particles
...

The inability to deliver the DNA systematically
...

The equipment itself is very expensive
...
Chemical methods:
1
...
The chemical used is
polyethylene glycol
...

Process:
The transfer of gene across the protoplast membrane can be initiated by a number of chemicals of which
polyethylene glycol is the most important
...

↓
After exposure of protoplasts to exogenous DNA in presence of PEG and other chemicals, PEG is removed
and intact protoplast are then cultured to form cells with walls and colonies in turn
...


2
...

It is called lipofection
...

Process:
Lipid solution + DNA
↓
Mix lipid and DNA
↓
DNA is encapsulated inside liposomes
↓
Liposome adheres to cell
↓
Liposome bilayer fuses with plasma membrane
↓
DNA enters cell
↓
DNA in nucleus
↓
Foreign gene expression

3
...
DAE-dextrin may be
used in the transfection medium in which DNA is present
...


By calcium phosphate: The process of transfection involves the admixture of isolated DNA with
solution of CaCl2 and potassium phosphate under condition which allows the precipitate of Ca 3(PO4)2
(calcium phosphate) to be formed
...
A fraction of cells will take up the calcium phosphate DNA precipitate by
endocytosis
...
Biological methods:
1
...

↓
Selection medium only allows plant cells that have acquired DNA from the bacteria to proliferate
...

2
...
It has some
limitations like that vast majority of plant viruses have genome not of DNA but RNA
...

Caulimoviruses: They contain circular dsDNA and are spherical in shape
...

Cauliflower mosaic virus (CaMV) infects many plants and can be easily transmitted
...
Its genome does not contain any non-coding regions
...

CaMV vectors has a limited capacity for insertion of foreign genes
...

Infective capacity of CaMV is lost if more than a few hundred nucleotides are
introduced
...

Herpes viruses cannot be used since the foreign DNA gets expelled and wildtype viruses are produced
...
They are particularly interesting
because their natural host include plant size such as maize and wheat
...


37

8
...
Gene Therapy
Gene therapy: Gene therapy is the process of inserting genes into cells to treat diseases
...
Thus, gene therapy primarily
involves genetic manipulations in animals or humans to correct a disease, and keep the organism in good health
...
Obviously, the goal of the
researchers is to benefit the mankind and improve their health
...
Somatic cell gene therapy: The non-reproductive (non-sex) cells of an organism are referred to as somatic

2
...
These are the cells of an organism other than sperm or egg cells e
...
bone marrow cells, blood cells,
skin cells, intestinal cells
...
In essence, somatic cell gene therapy involves the insertion of a fully functional
and expressible gene into a target somatic cell to correct a genetic disease permanently
...
Gene
therapy involving the introduction of DNA into germ cells is passed on to the successive generations
...


The genetic alterations in somatic cells are not carried to the next generations
...

Steps in gene therapy:

1
...

3
...


In vivo experiments and research on laboratory animals (pre-clinical trials)
...

Phase II trials with more human subjects to assess whether the product is helpful
...


As such, gene therapy involves a great risk
...
Recombinant DNA Advisory Committee (RAC) is the
supervisory body of the National Institute of Health, USA that clears proposals on experiments involving gene
therapy
...

Types: There are two basic types of gene therapy:

1
...

Steps:
Isolation of totipotent embryonic cells at an undifferentiated stage
↓
The determination of the genetic state of the embryo
↓
Embryonic stem cell expansion in culture
↓
Transfer of the genetic material into embryonic cell
↓
Selection of stably transfected cell
↓
Targeted gene replacement
↓
Removal of marker
↓
Confirmation of genomic integrity
↓

39

Transfer of the nucleus
↓
Re-implantation in the mother

2
...
It is further divided into two types:
i
...
g
...

Steps:
Isolation of cell with genetic defect from a patient
...

↓
Introduction to the therapeutic gene to correct gene defect
...

↓
Transplantation of the modified cells to the patient
...
This technique, is therefore, not associated with adverse
immunological responses after transplanting the cells
...
This can be
achieved by using vectors
...
Therapy for adenosine deaminase (ADA) deficiency
2
...
Therapy for Lesch-Nyhan syndrome
ii
...

Process:
Copies of the therapeutic gene are inserted into viral DNA, liposome or in the form of plasmid
DNA
↓
Genetically altered DNA is inserted into patient’s body by cell specific direct tissue injection
...
These cells now encode and produce the needed protein encoded by the inserted gene
...
The efficiency of the uptake of the remedial (therapeutic) gene by the target cells
...
Intracellular degradation of the gene and its uptake by nucleus
...
The expression capability of the gene
...
Viral vectors and
B
...
Viral vectors:
1
...
Non-viral vectors: It may be chemical or physical
...
Chemical:
• Nanoparticles
• Polyplexes
• Polymeric micelles
• Lipoplexes
b
...

3
...

5
...
Gene transfer is more efficient and specific than physical and chemical methods
...
Multiple and repeated doses are required in case of physical and chemical method, whereas in case of viral
vector even a single dose is sufficient
...

2
...

4
...

6
...

Repeated treatments are needed
...

Viral vectors could become pathogenic
...

Multigene disorders are too complex to treat
...

2
...

4
...

6
...

8
...


Curing genetic diseases
...

Inducing cancerous cells to make toxins so that they kill themselves
...

Gene therapy to enhance cancer treatment
...

For the treatment of neurological disorder
...
g
...

Creating stem cells from somatic cells
...
Gene therapy clinical trials for cancer are moving rapidly from phase I to III and the first anti-cancer gene
2
...

4
...

Some serious adverse events following gene therapy have been occurred, but progress has been made in
understanding and overcoming these problems
...

Ex vivo transduction of stem cells with integrating retroviruses ensures high and persistent level of gene
transfer and is promising strategy for several diseases
...
Lentiviral vectors will move from preclinical safety assessment studies to clinical trials
...
Studies to evaluate the genotoxicity of any gene transfer vector will become a fundamental feature of gene
3
...

Stem cells from a range of different tissues or organs will become the target cells for many gene therapy
applications
...
Unwanted immune system reaction: Body’s immune system may see the newly introduced viruses as
2
...

4
...
This may cause inflammation and in severe cases, organ failure
...
If this
happens, healthy cells may be damaged, causing other illness or diseases such as cancer
...

Possibility of causing a tumor: If the new genes get inserted in the wrong spot in the DNA, there is a
chance that the insertion might lead to tumor formation
...
It is often used in combination with other fertility
treatments such as in vitro fertilization and intrauterine instrumentation, to increase a woman’s chance of being
pregnant successfully
...
It is also called controlled ovarian
stimulation
...
The goal of biomedical engineering is to use electrical, chemical
and mechanical engineering principles to conduct studies and develop tools that can aid in the biomedical care of
patients
...

2
...

4
...

6
...

8
...

10
...


Biomechanics
Prosthetic devices and artificial organs
Transport phenomena
Biomaterials
Biomedical instrumentation
Biosensors
Medical and biologic analysis
Medical imaging
Biotechnology
Clinical engineering
Medical informatics

RFLP
RFLP (restriction fragment length polymorphism): It is the inherited difference in the pattern of restriction
fragments among the individuals caused by insight variation in the base sequence of their DNA
...

2
...


Single base change or single nucleotide polymorphism (SNP)
...

VNTR (variable number of tandem repeats)

Importance:
1
...


It serves as the basis of DNA fingerprint for person identification at molecular level
...


Genetic polymorphism or polymorphism
Polymorphism: It is the variation in DNA sequence from one individual to another having no impact on health
and functioning of gene (no phenotypic effect)
...

Synonym: Genetic diversity or variability
Mechanism: DNA sequence variation occurs by –
1
...

3
...


43

Ti plasmid
Ti plasmid is the large sized tumor inducing plasmid found in Agrobacterium tumefaciens
...
A
...
They infect plants through breaks or wounds
...

Characteristics:
1
...

3
...


A Ti or tumor inducing plasmid is a plasmid that often, but not always, is a part of the genetic equipment
that A
...
rhizogenes use to transduce its genetic material to plants
...

The plasmid has 196 genes that code for 195 proteins
...

The modification of this plasmid is very important in the creation of transgenic plants
...

2
...


T DNA region: This region has the genes for the biosynthesis of auxin (aux), cytokinin (cyt) and opine
(ocs) and is flanked by left and right borders
...

Opine catabolism region: This region codes for proteins in the uptake and metabolism of opines
...
tumefaciens
...

2
...

4
...
Smaller vectors are preferred for recombinant experiment
...

The phytohormones (auxin and cytokinin) produced by the plant cells
...

Opine production in transformed plant cells lowers the plant yield, so opine synthesizing genes should be
removed
...

Steps of chromosome walking:
From the genomic library select a clone of interest and subclone a small fragment from one end of the clone
↓
The subcloned fragment of the selected clone may be hybridized with other clones in the library and a second clone
hybridizing with the subclone of the first clone is identified due to presence of overlapping region
↓
The end of the second clone is then sucloned and used for hybridization with other clones to identify a third clone
having overlapping region with the subcloned end of the second clone
↓
Third clone identified as above is also subcloned and hybridized with clones in the same manner and the procedure
may continued
↓
Restriction map of each selected clone may be prepared and compared to know the regions of overlapping so that
the identification of new overlapping restriction sites will amount to walking along the chromosome or along a
long chromosome segment
...

Basic chemistry:
Nucleotide = Nitrogenous base + Pentose sugar + Phosphate
Nucleoside = Nitrogenous base + Pentose sugar
Types:
1
...


Purine e
...
adenine and guanine
...
g
...


Functions or biomedical importance:
1
...

3
...

5
...

7
...


They are building blocks or monomers of nucleic acids
...

Many coenzymes are derivatives of nucleotides e
...
NAD+ , NADP+ etc
...
g
...

They play an important role as energy currency in the cell
...
g
...

ADP level regulates oxidative phosphorylation
...


45

Short Notes
Codon
The sequence of bases in the DNA of living cells that provides the instructions for the synthesis of proteins from
amino acids is called genetic code
...

Properties:
1
...

3
...
e
...

Each codon represents a specific amino acid
...
There are 43 = 64 codons
...

2
...

Termination (stop or nonsense) codons: Three of the 64 possible codons that do not code for specific
amino acids; they are called nonsense or termination codons
...
When
one of these codons appears in an mRNA sequence, it signals that the synthesis of the peptide chain is
completed
...

Types of genes:
1
...

3
...

5
...


Dominant genes
Co-dominant genes
Recessive genes
Carrier genes
Sex linked genes
Sex limited genes

Structure of a gene: Genes are mosaics of exons and introns flanked by promoter, terminator, enhancer and
silencer
...

2
...


4
...

6
...

Introns (non-coding segment): This is the sequence of gene that is transcribed but excised before
translation
...
Introns are always removed from the
precursor RNA before transportation of the cytoplasm occurs
...

TATA or Hogness box, CAAT box, GC box are promoters of eukaryotic genes
...

Terminator: Sequence in the downstream direction is called terminator
...

Silencer: It lies in the upstream, downstream or within gene
...

Enhancer: It lies in the upstream, downstream or within gene
...


Ion exchange chromatography
Ion exchange chromatography is a subset of liquid chromatography which is a process that allows the separation
of ions and polar molecules based on their charge
...

Process:

46

1
...
This process of eluent ion (E⸺) displacing an anion (X⸺) bonded to the resin can be
expressed by the following chemical interaction:
Resin+ ⸺ X⸺ + E⸺ ↔

2
...


4
...
This sample could contain many
different ions, but for simplicity this example uses just two different ions as analytes in the sample
...
As
the sample elutes (or moves through the column), anion A and anion B adhere to the column surface
differently
...

As the eluent continues to be added, the anion A moves through the column in a band and ultimately is
eluted first
...

Resin+ ⸺ A⸺ + E⸺ ↔

5
...

Resin+ ⸺ B⸺ + E⸺ ↔

Resin+⸺E⸺ + B⸺

[Elution is the process where the compound of interest is moved through the column
...

Uses:
1
...

3
...

5
...


Operon
According to operon model, proposed by F
...
Monod, an operon was defined as a unit of coordinated
control of protein synthesis, which consisted of (i) an operator gene which controlled the activity of (ii) a number
of structural genes which took part in the synthesis of protein(s)
...
The operator gene, in turn, is under the control of a
repressor molecule synthesized by a regulator gene, which is not a part of the operon
...
One such a operon in E
...

The lac operon system is describe hereunder:
Structure: A lac operon contains three structural genes or cistrons namely z, y and a, whose products (enzymes)
are involved in the breakdown of the sugar lactose
...
Gene a codes for thiogalactoside acetylase, which transfers an acyl
group from acetyl Co A to β galactoside (i
...
this enzyme is indirectly involved in lactose utilization)
...

Mechanism:
In the absence of lactose (inducer), the regulator gene produces a repressor protein, which binds to the operator site
and prevents the transcription
...

In the absence of lactose, it binds to the repressor protein; the repressor now fails to bind to the operator
...
This mRNA codes for three enzymes necessary for lactose catabolism
...
This permanent state entails benefits and
detriments for the organism in which the cells live
...

Characteristics of senescent cells:
1
...

3
...

They are larger than normal cells
...


Causes of cellular senescence:
1
...


3
...
Telomeres
are sequences of DNA that are found at each end of the chromosome
...
These repetitions protect the chromosome from losing important
information during replication and from using with nearby chromosome
...
Thus, the chromosomes are shortened after each replication until they reach a point
at which, after having lost the telomere, they lose important genetic information
...

Because this is a natural process of aging, more cells become senescent as we grow older
...

Other types of DNA damage: Other types of DNA damage can also induce cellular senescence by
damaging DNA
...

Other factors: These include –
i
...
g
...

ii
...
g
...

iii
...
g
...


Effects of cellular senescence:
1
...

3
...

In older individuals, it induces the spread of tumors
...


Pedigree chart
A pedigree chart is a diagram that shows the occurrence and appearance of phenotypes of a particular gene or
organism and its ancestors from one generation to the next
...

2
...

4
...

6
...

Pedigrees use a standardized set of symbols
...

Relationships in a pedigree are shown as a series of lines
...

Analysis of a pedigree using the principles of Mendelain inheritance can determine whether a trait has a
dominant or recessive pattern of inheritance
...

2
...

4
...


A pedigree may be used to establish the probability of a child having particular disorder or condition
...

Determination of whether a trait is dominant or recessive
...

When the condition predominantly affects males in the pedigree, it is considered X linked
...
It is a non-ionic detergent, which forms insoluble complexes
with nucleic acids if the sodium chloride concentration in the solution is around 0
...
It is used for the preparation
of total cell DNA from plants
...

↓
The precipitate is then collected by centrifugation and re-suspended in 1M solution of chloride, which causes the
complex to break down
...

RT PCR
Reverse transcriptase PCR (RT PCR) was developed to amplify RNA targets (RNA viruses such as HIV, HCV and
influenza are key examples)
...

Principle: PCR depends on the Taq DNA polymerase enzyme; RNA is not an efficient substrate for this enzyme
...

Process:
After RNA is released from cellular material through extraction; an aliquot of the extracted sample is added to a
reaction mixture which contains reverse transcriptase enzyme, primers specific for the target of interest and
nucleotides
...

↓

49

Reverse transcriptase enzyme synthesizes a complementary DNA strand, extending from a primer
...

↓
The temperature is lowered to about 650 C allowing primers to anneal to the newly formed cDNA
...

↓
Those steps are repeated over and over, so the number of DNA molecules increases exponentially
...

2
...

4
...


RT PCR is commonly used to study the genome of viruses whose genomes are composed of RNA, such
as HIV and influenza
...
This helps early and confirmatory diagnosis of
infectious diseases like AIDS, TB etc
...

Prenatal diagnosis of genetic diseases e
...
cystic fibrosis
...


Cystic fibrosis
Cystic fibrosis is an inherited disorder that causes severe damage to the lungs, digestive system and other organs in
the body
...

Cause: In cystic fibrosis, a defect (mutation) in a gene changes a protein that regulates the movement of salt in and
out of the cells
...


Symptoms:
1
...

3
...

5
...

7
...


A persistent cough that produces thick mucus
...

Exercise intolerance
...

Inflamed nasal passages or a stuffy nose
...

Intestinal blockage, particularly in newborns
...


Risk factors:
1
...


Family history: Because cystic fibrosis is an inherited disorder, it runs in families
...


Therapeutic proteins
Therapeutic proteins are proteins that are engineered in the laboratory for pharmaceutical use
...
Protein therapeutics permit an
individualized treatment approach by supporting a specifically targeted therapeutic process by compensating the
deficiency of an essential protein
...

2
...


Production of therapeutic proteins in microbial bioreactors e
...
E
...

Mammalian cell derived bioreactors e
...
Chinese Hamster Ovary cell (CHO) bioreactors
...
g
...


Applications:
1
...

3
...

5
...

7
...


Used in medical applications
...

Interferons
...

Recombinant protein vaccines
...

Biological response modifiers
...


Advantages:
1
...

3
...

5
...


They have a highly specific and complex set of functions
...

Well tolerated and less likely to elicit immune responses
...

A faster clinical development and FDA approval
...


Meiosis
Meiosis is a process where a single cell divides twice to produce four cells containing half of the original amount of
genetic information
...

Process:
A
...


Prophase I: The homologous chromosomes pair and exchange DNA to form recombinant
chromosomes
...


Leptotene: chromosomes start to condense
...

Pachytene: crossing over between pairs of homologous chromosomes to form chiasmata
(sing
...

Diplotene: homologous chromosomes start to separate but remain attached by chiasmata
...


Pro-metaphase I: Spindle apparatus formed and chromosomes attached to spindle fibers by
kinetochores
...


Metaphase I: Homologous pairs of chromosomes (bivalents) arranged as a double row along the
metaphase plate
...


Anaphase I: The homologous chromosome in each bivalent are separated and the nuclear membrane
reforms
...


Cytokinesis: The final cellular division to form two new cells, followed by meiosis II
...
Meiosis II: Meiosis II separates each chromosome into two chromatids
...


Prophase II: Chromosomes begin to condense, nuclear membrane dissolves and spindle fibers
form
...


Metaphase II: Spindle fibers attach to chromosomes
...


3
...


4
...


5
...


Importance: Meiosis generates genomic diversity through –
1
...


2
...


3
...


Agrobacterium
Agrobacterium tumefaciens is a Gram-negative soil bacterium, which naturally transforms plant cells, resulting in
crown gall (cancer) tumors
...
Tumor formation is the result of T-DNA
(transfer DNA) in plant genome
...
The process is as follows:
Discs removed from tobacco leaf
↓
Leaf discs incubated with genetically engineered Agrobacteria for 24 hours
...

↓
Transfer to shoot inducing medium
↓
Transfer shoot to root-inducing medium
↓
Rooted seedlings are grown up
↓
Adult plant carrying transgene that was originally present in bacteria

---