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Unit I

B. Tech. (Biotechnology) III Year


VI th Semester

TBT-604, Genetic Engineering


Unit I
• Gene cloning -concept and basic steps
• Application of bacteria and viruses in genetic
engineering
• Molecular biology of E. coli and bacteriophages
in the context of their use in genetic engineering
• Cloning vectors: Plasmid cloning vector PBR322,
• Vectors for cloning large piece of DNA
– Bacteriophage-l and other phage vectors
– Cosmids
– Phagemids
– YAC and BAC vectors
• Model vectors for eukaryotes - Viruses,
Three Great Milestones in
Genetics
• Gregor Mendel: the rules of inheritance
• James Watson and Francis Crick: the
structure of DNA
• The Human Genome Project: the detailed
analysis of human DNA
Mendel: Genes and the
Rules of Inheritance (1866)
• Genes—hereditary
factors responsible for
traits
• Alleles—different forms
of genes
• Rules of Inheritance
– Alleles of the same gene
separate during gamete
formation
– Alleles of different genes
are inherited independently
What is a Gene?

• Genes are made of nucleic acids


• Nucleic acids are made of building
blocks called nucleotides
• Nucleotides have three
components
– Sugar molecule (ribose or
deoxyribose)
– Phosphate molecule
– Nitrogen-containing molecule
(adenine, guanine, cytosine,
thymine, uracil)
• RNA is ribonucleic acid
• DNA is deoxyribonucleic acid
Watson and Crick:
The Structure of DNA (1953)
• Nucleotides are linked in
a chain through sugar-
phosphate interactions
• DNA molecules are made
of two chains of
nucleotides wound
around each other in a
helix
• Base pairs hold the
chains together
– A pairs with T
– G pairs with C
The Human Genome Project:
Sequencing DNA and Cataloguing
Genes
• Genome—the collection of
DNA molecules that is
characteristic of an organism
• Genomics is the analysis of
DNA sequences that make up
a genome
• Genomics involves DNA
sequencing technology,
robotics, and computer
science
• The Human Genome Project
determined the sequence of
nucleotides in the DNA of the
human genome
A Milestone in Genetics:
φ X174, the First DNA Genome Sequenced
∀ φ X174 is a virus that
has single-stranded
DNA as its genetic
material.
• Frederick Sanger
sequenced the
genome of φ X174 in
1977
DNA as the Genetic Material
• Information flows from DNA to RNA to
protein.
• In all cellular organisms, the genetic
material is DNA.
• The genetic material
– Must be able to replicate
– Must contain information
– Must be able to change
Gene Expression:
Using Genetic Information
Gene Expression
• During transcription, an RNA molecule is
synthesized from a DNA template.
• This messenger RNA (mRNA) molecules
contains the information needed to synthesize a
polypeptide.
• During translation, the triplet codons in the
RNA specify the incorporation of particular
amino acids into a polypeptide chain.
The Proteome
• Proteome—the collection of all the
different proteins in an organism.
• Humans have between 20,000 and 25,000
genes in the genome and hundreds of
thousands of proteins in the proteome.
• Proteomics—the study of all the proteins
in cells.
The Central Dogma of
Molecular Biology

• The flow of information is DNA  RNA protein.


• Some viruses can use RNA as a template for the
synthesis of DNA in reverse transcription.
• Many genes do not encode polypeptides; their end-
products are RNA molecules.
Classical Genetics
• Based on analysis of the outcomes of crosses
between different strains of organisms.
• Can be coordinated with studies of the structure
and behavior of chromosomes.
• Encompasses transmission genetics and
studies of the nature of the genetic material
Molecular Genetics
• Studies the replication, expression, and
mutation of genes at the molecular level.
• Rooted in the study of DNA sequences
and the manipulation of DNA molecules.
Gene cloning -concept and basic steps

Recombinant DNA, gene cloning, and


DNA amplification techniques allow
scientists to isolate and characterize
essentially any DNA sequence from
any organism.
Gene Cloning
• Gene cloning is the isolation and
amplification of a given gene.

• A recombinant DNA molecule is a DNA


molecule made by joining two or more
different DNA molecules.
What is Gene Manipulation?
Creation and cloning of r-DNA

• r-DNA
– Artificially created DNA molecule which bring together
DNA Sequences not usually found together in nature
• Gene Manipulation
– Any, of variety of sophisticated techniques for creation
of r-DNA and (in many cases) its subsequent
introduction in living cell
• Cloning
– Propagation of r-DNA inside a particular host, so that
many copies of same sequence are produced
Amplification of a Gene In Vivo
• A minichromosome carrying the gene of
interest is produced in the test tube.
• The recombinant minichromosome is
introduced into a host cell (such as E.
coli), and the host cell replicates the
minochromosome.
Amplification of a Gene In Vitro
• Short DNA strands complementary to
DNA sequences on either side of the gene
of interest are synthesized.
• These short DNA strands are used to
initiate the amplification of the gene by a
heat-stable DNA polymerase in the
polymerase chain reaction (PCR).
Vectors
• Plasmid Vectors
• Bacteriophage Vectors
• Cosmid Vectors
• Shuttle Vectors
• Eukaryotic Vectors
• Yeast Artificial Chromosomes (YACs)
• Bacterial artificial chromosomes (BACs)
• Bacteriophage P1 artificial chromosomes
(PACs)
Plasmid Vectors
• Circular, double-stranded circular DNA
molecules present in bacteria.
• Range from 1 kb to over 200 kb.
• Replicate autonomously.
• Many carry antibiotic-resistance genes, which
can be used as selectable markers.
• Many useful cloning vectors were derived from
plasmid pBR322.
Plasmid Vectors
• A plasmid is a genetic element that can replicate
independently of the main chromosome in an
extrachromosomal state.
• Most plasmids are not required for the survival of
the host cell.
• Plasmids in E. coli
– F Factor (Fertility Factor)
– R Plasmids (Resistance Plasmids)
– Col Plasmids (synthesize compounds that kill
sensitive cells)
Features of many modern Plasmids

•Small size
•Origin of replication
•Multiple cloning site (MCS)
•Selectable marker genes
•Some are expression vectors and have sequences
that allow RNA polymerase to transcribe genes
•DNA sequencing primers
Essential Features of a Cloning
Vector
• Origin of replication
– essential for self-
replication in host
cells
• Dominant selectable
marker gene
– usually confers drug
resistance
• One or more unique
restriction sites
A Polycloning Site is a Cluster
of Unique Restriction Sites
Episomes
• An episome is a genetic element that is
not essential to the host and that can
either replicate autonomously or be
integrated into the bacterial chromosome.
• Integration depends on the presence of IS
elements.
Bacteriophage Vectors

• Most bacteriophage cloning vectors have


been constructed from the phage λ
chromosome.
• The central one-third (about 15 kb) of the λ
chromosome contains genes required for
lysogeny but not for lytic growth.
• This portion of the chromosome can be
excised and replaced with foreign DNA.
• The foreign DNA inserted must be 10-15 kb.
Cosmid Vectors
• Hybrids between
plasmids and the phage
λ chromosome.
• Replicate autonomously
in E. coli.
• Can be packaged in vitro
into phage λ heads.
• Accept inserts of 35-45
kb.
Phagemid Vectors
• Contain components from phage chromosomes
and plasmids.
• Replicate in E. coli as double-stranded plasmids.
• Addition of a helper phage causes the phagemid
to switch to the phage mode of replication,
resulting in the packaging of single-stranded
DNA into phage heads.
The Life Cycle of Bacteriophage
M13
Phagemids pUC118 and pUC119
Replication as Double-Stranded
Plasmids
Replication as Single-Stranded
Phage DNA
The Blue-White Color Test
• The E. coli lacZ gene
encodes β -galactosidase.
∀ β -galactosidase converts
the colorless substrate Xgal
into a blue product.
• Cells with β -galactosidase
activity produce blue colonies
when grown on Xgal; cells
lacking β -galactosidase
activity produce white
colonies.
Eukaryotic and Shuttle Vectors
• Because different organisms use different
origins of replication and regulatory signals,
different cloning vectors must be used in
different species.

• Special cloning vectors can replicate in other


prokaryotes and in eukaryotes.

• Shuttle vectors can replicate in E. coli and in


another species.
An E. coli-Yeast Shuttle Vector
Yeast Artificial Chromosomes (YACs)

• Genetically engineered yeast minichromosomes.


• Accept foreign DNA inserts of 200-500 kb.
• Contain a yeast origin of replication, yeast centromere,
two yeast telomeres, a selectable marker, and a
polycloning site.
BACs and PACs
• Bacterial artificial chromosomes (BACs) have
been constructed from bacterial fertility (F)
factors.
• Bacteriophage P1 artificial chromosomes
(PACs) have been constructed from
bacteriophage P1 chromosomes.
• BACs and PACs accept 150-300 kb inserts and
are less complex than YACs.
The PAC Mammalian Shuttle
Vector pJCPAC-Mam1
Restriction Endonucleases
• Restriction endonucleases make site-specific
cuts in DNA.
• The nucleotide sequences are called restriction
sites.
• Restriction endonucleases protect bacteria from
foreign DNA.
• Bacteria protect endogenous restriction sites by
methylation.
• Restriction enzymes commonly recognize
palindromic sequences.
Structure of an EcoRI-DNA
Complex
Many Restriction Endonucleases
Make Staggered Cuts
• When DNA is cleaved with a restriction
endonuclease that makes staggered cuts, all
of the resulting restriction fragments have
complementary single-stranded termini.

• The complementary single-stranded termini


can hydrogen bond with each other and be
joined together by DNA ligase.
Construction of Recombinant
DNA Molecules In Vitro