Cloning and Analysis of Cloned Genes

Term cloning

Producing genetically identical copies of an organism, cell, or DNA sequence; may refer to whole-organism cloning (e.g., Dolly the sheep) or molecular cloning of DNA fragments.

Gene cloning

Producing many identical copies of a specific segment of DNA using vectors and host cells.

Difference between organism cloning and gene cloning

Organism cloning replicates an entire organism; gene cloning replicates only a DNA fragment.

Why clone DNA?

To study gene structure, sequences, control regions, protein/RNA functions, mutations, and engineer organisms for research or therapy.

Benefits of DNA cloning

Improved diagnostics, disease understanding, therapy development, drug production, crop/animal engineering, environmental applications.

Example: TPA in milk

TPA gene expressed under β-lactoglobulin promoter in transgenic sheep to produce clot-dissolving protein in milk.

Why replicate a single DNA fragment?

To obtain unlimited pure copies for analysis, sequencing, or expression.

Need for cutting chromosomes

Large chromosomes cannot be cloned directly; restriction enzymes cut them into gene-sized fragments.

Restriction enzymes

Proteins that recognize specific DNA sequences and cut DNA to create useful fragments.

Basic cloning tools

Restriction enzymes, vectors, ligase, transformation, antibiotic selection.

Cloning vector

DNA molecule with an origin of replication that carries foreign DNA and replicates in host cells.

Plasmid

Small circular DNA in bacteria capable of independent replication; common cloning vector.

Plasmid vector components

Origin of replication, antibiotic resistance gene, multiple cloning site (MCS).

Types of vectors

Plasmids (small inserts), cosmids, BACs (large inserts), YACs (very large inserts).

F-plasmid significance

Derived backbone for BACs; stable replication and maintenance of large inserts.

Recombinant DNA

DNA made by ligating vector DNA and foreign DNA with ligase.

DNA ligase

Enzyme joining DNA fragments by sealing sugar-phosphate backbone.

Transformation

Introduction of plasmid DNA into bacteria.

Competent cells

Bacteria treated to allow DNA uptake (often with CaCl₂).

Role of CaCl₂

Neutralizes negative charges, makes membrane brittle, helps DNA bind surface.

Purpose of heat shock

Creates temporary membrane opening so DNA enters the cell.

Transformation outcome

Only bacteria with plasmid survive antibiotic selection; each colony carries plasmid copies.

Steps in DNA cloning

Digest DNA → separate fragments → ligate → transform → select → screen.

Possible ligation outcomes

Vector only, insert only, or vector+insert (desired recombinant).

Colony screening

Identifies bacteria with recombinant plasmids using antibiotics and restriction digestion.

Gene expression

Production of protein from DNA; requires promoter, signals, and host system.

Expression vs cloning vectors

Expression vectors have promoter + poly(A); cloning vectors do not.

Transcription cassette

Promoter + cloned gene + poly(A) signal.

Reporter gene

Easily measurable gene (GFP, luciferase, CAT) used to test regulatory sequences.

Why introns block bacterial expression

Bacteria cannot remove introns; eukaryotic genes must be intron-free to be expressed.

Reverse transcriptase

Enzyme that converts mRNA into intron-free cDNA.

cDNA cloning

Creating DNA copies of mature mRNA to clone functional genes.

Transformation vs transfection

Transformation = bacteria; transfection = eukaryotic cells.

In vitro expression systems

Expression in mammalian, bacterial, yeast, or insect cells.

Modified expression vectors

Contain organism-specific promoters and terminators for expression.

Transgenic animals

Animals engineered to express foreign genes for physiological studies.

Microinjection technique

Inject DNA into fertilized egg pronucleus; integrates randomly.

Problems with microinjection

Low efficiency, random insertion, low expression.

Embryonic stem cell technology

Uses homologous recombination to target gene insertion; ES cells injected into blastocysts.

mRNA analysis methods

RT-PCR, real-time PCR, Northern blot, in situ hybridization.

Protein analysis methods

Western blot, ELISA.

Safety concerns

Long-term effects, environmental spread, new pathogens, antibiotic resistance spread.

Ethical issues

Gene patenting, profit ownership, genetic privacy, screening, designer babies.

GMO concerns

Food safety, ecological effects, gene flow, regulation.

Genome-edited babies

Ethical issues around heritable human gene modification.

Legislative considerations

Questions of who controls genetic manipulation: individuals, institutions, or governments.