In-Depth Notes on Recombinant DNA Technology

Recombinant DNA Technology

• Overview: Involves the joining of DNA from different sources, typically in a laboratory setting. This technology allows for various applications, including cloning, gene expression study, and genetic modifications.

Key Concepts

• Cloning: The process of creating identical DNA molecules, cells, or organisms from an original source. It involves the following:

  • Use of cloning vectors: DNA molecules, such as plasmids, that can accept DNA fragments and replicate within a host cell.

  • Criteria for cloning vectors:

  1. Contains restriction cut sites for DNA insertion.

  2. Ability to replicate in a host cell.

  3. Incorporates selectable marker genes and/or reporter genes.

  4. Contains sequences for DNA fragment sequencing.

Restriction Enzymes

• Function: Cut DNA at specific sequences, called palindromic recognition sites.

  • Types of cuts:

  • Blunt ends: Cuts are at the same position on both strands.

  • Sticky ends: Cuts are at different positions, allowing for easier reannealing due to complementary base pairing.

• Applications: Use of ligase to seal nicks and create recombinant DNA by mixing DNA from different sources cut with the same restriction enzyme.

Transformation

• Definition: The process of introducing plasmids into bacterial cells. Two main methods:

  1. Heat-shock method: Cells are mixed with plasmids and briefly heated to create pores in the cell membrane, allowing plasmids to enter.

  2. Electroporation: Electric shock is applied to a mixture of cells and plasmids, creating pores for plasmid entry.

Selectable Markers and Reporter Genes

• Selectable Marker: A gene that allows for selection of transformed cells, such as antibiotic resistance genes. Indicates the presence and expression of gene. For example:

  • Cells containing plasmids with ampicillin resistance can grow on media with ampicillin.

• Reporter Gene: Indicates the presence and expression of a gene through visible phenotypes. If plasmid is not recombinant, reporter gene is expressed. If plasmid is recombinant, reporter gene is disrupted and is not expressed. Example:

  • LacZ gene: Produces β-galactosidase, which turns the media blue when active (indicating non-recombinant plasmids) and remains white when disrupted (indicating recombinant plasmids).

DNA Libraries

• Definition: Collections of DNA fragments from a genome placed in vectors and transformed into bacteria.

  • Genomic Library: Contains all genomic DNA from an organism, including coding and non-coding regions.

  • cDNA Library: Contains only expressed genes; mRNA is reverse-transcribed into cDNA, allowing the study of gene expression.

PCR (Polymerase Chain Reaction)

• Function: Amplifies a specific DNA segment in vitro, significantly faster than traditional cloning.

• Components:

  • DNA polymerase: Heat-tolerant enzyme that synthesizes new DNA strands.

  • dNTPs: Building blocks for new DNA strands.

  • Primers: Short single-stranded DNA sequences that flank the target region and provide starting points for DNA synthesis.

PCR Steps**

1. Denaturation: Heating the DNA to separate it into single strands (95°C).

2. Annealing: Lowering the temperature to allow primers to bind (45-65°C).

3. Extension: DNA polymerase synthesizes new DNA strands starting from the primers (72°C).

• The cycle is repeated 30 times, resulting in millions of copies of the target DNA.

Applications of Recombinant DNA Technology

• Pharmaceutical Production: Generating insulin and vaccines through genetically modified organisms.

• Gene Therapy: Treating diseases by correcting defective genes.

• Forensic Science: PCR used for DNA fingerprinting and evidence analysis.

• Paleontology and Archaeology: Recovering ancient DNA for study.

Variations of PCR**

• RT-PCR: Converts mRNA to cDNA to study gene expression.

• qRT-PCR: Quantitative real-time PCR measures how much a gene is expressed using fluorescent probes.

Recombinant DNA Technology - Overview: Involves the joining of DNA from different sources, typically in a laboratory setting. This technology allows for various applications, including cloning, gene expression study, and genetic modifications.

Key Concepts - Cloning: The process of creating identical DNA molecules, cells, or organisms from an original source. It involves the following:

• Use of cloning vectors: DNA molecules, such as plasmids, that can accept DNA fragments and replicate within a host cell.

• Criteria for cloning vectors:

  1. Contains restriction cut sites for DNA insertion.

  2. Ability to replicate in a host cell.

  3. Incorporates selectable marker genes and/or reporter genes.

  4. Contains sequences for DNA fragment sequencing.

• YACs (Yeast Artificial Chromosomes): Vectors that can carry large fragments of DNA, up to 1 million base pairs, and can be used in the cloning process, especially for genomic libraries.

• BACs (Bacterial Artificial Chromosomes): Another type of large vector that can carry up to 300,000 base pairs of DNA, useful in mapping and sequencing genomes.

Restriction Enzymes - Function: Cut DNA at specific sequences, called palindromic recognition sites.

• Types of cuts:

  • Blunt ends: Cuts are at the same position on both strands.

  • Sticky ends: Cuts are at different positions, allowing for easier reannealing due to complementary base pairing.

• Applications: Use of ligase to seal nicks and create recombinant DNA by mixing DNA from different sources cut with the same restriction enzyme.

Transformation - Definition: The process of introducing plasmids into bacterial cells. Two main methods:

1. Heat-shock method: Cells are mixed with plasmids and briefly heated to create pores in the cell membrane, allowing plasmids to enter.

2. Electroporation: Electric shock is applied to a mixture of cells and plasmids, creating pores for plasmid entry.

Selectable Markers and Reporter Genes - Selectable Marker: A gene that allows for selection of transformed cells, such as antibiotic resistance genes. Indicates the presence and expression of gene. For example:

• Cells containing plasmids with ampicillin resistance can grow on media with ampicillin.

• Reporter Gene: Indicates the presence and expression of a gene through visible phenotypes. If plasmid is not recombinant, reporter gene is expressed. If plasmid is recombinant, reporter gene is disrupted and is not expressed. Example:

• LacZ gene: Produces β-galactosidase, which turns the media blue when active (indicating non-recombinant plasmids) and remains white when disrupted (indicating recombinant plasmids).

DNA Libraries - Definition: Collections of DNA fragments from a genome placed in vectors and transformed into bacteria.

• Genomic Library: Contains all genomic DNA from an organism, including coding and non-coding regions.

• cDNA Library: Contains only expressed genes; mRNA is reverse-transcribed into cDNA, allowing the study of gene expression.

PCR (Polymerase Chain Reaction) - Function: Amplifies a specific DNA segment in vitro, significantly faster than traditional cloning.

• Components:

  • DNA polymerase: Heat-tolerant enzyme that synthesizes new DNA strands.

  • dNTPs: Building blocks for new DNA strands.

  • Primers: Short single-stranded DNA sequences that flank the target region and provide starting points for DNA synthesis.

PCR Steps** 1. Denaturation: Heating the DNA to separate it into single strands (95°C).

2. Annealing: Lowering the temperature to allow primers to bind (45-65°C).

3. Extension: DNA polymerase synthesizes new DNA strands starting from the primers (72°C).

• The cycle is repeated 30 times, resulting in millions of copies of the target DNA.

Applications of Recombinant DNA Technology - Pharmaceutical Production: Generating insulin and vaccines through genetically modified organisms.

• Gene Therapy: Treating diseases by correcting defective genes.

• Forensic Science: PCR used for DNA fingerprinting and evidence analysis.

• Paleontology and Archaeology: Recovering ancient DNA for study.

Variations of PCR** - RT-PCR: Converts mRNA to cDNA to study gene expression.

• qRT-PCR: Quantitative real-time PCR measures how much a gene is expressed using fluorescent probes.