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 theRules 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:X174, the First DNA Genome Sequenced

X174 is a virus that has single-stranded DNA as its genetic material.

• Frederick Sanger sequenced the genome of 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)

•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

Features of many modern Plasmids

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 chromosome.

• The central one-third (about 15 kb) of the 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 chromosome.

• Replicate autonomously in E. coli.

• Can be packaged in vitro into phage 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 -galactosidase.

-galactosidase converts the colorless substrate Xgal into a blue product.

• Cells with -galactosidase activity produce blue colonies when grown on Xgal; cells lacking -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

Construction of Recombinant DNA Molecules In Vitro