DNA Cloning W3

Learning Outcomes

• Outline some of the ways in which genes may be isolated, cloned, sequenced, amplified, and expressed.

• Demonstrate an understanding of a range of modern molecular biology techniques such as PCR, DNA manipulation, and sequencing using traditional and current techniques.

Introduction to DNA Cloning

• DNA Cloning Definition: The production of multiple identical copies of a specific DNA molecule. This process typically involves isolating a target gene or DNA segment, inserting it into a suitable vector, and then introducing the recombinant vector into a host organism where it can be replicated.

• Requirement: Cloning requires a molecular toolkit, which includes:

  • Vectors: These are DNA molecules, such as plasmids or viruses, that can carry foreign DNA into a host cell and facilitate its replication. They provide the vehicle for DNA propagation.

  • Cutting Enzymes (DNA Restriction Endonucleases): These are enzymes that act as "molecular scissors", specifically recognizing and cutting double-stranded DNA at precise nucleotide sequences (recognition sites). These cuts create fragments that can be inserted into vectors.

  • Pasting Enzymes (DNA Ligases): These enzymes act as "molecular glue", forming phosphodiester bonds between DNA fragments, thereby joining them together. This is crucial for inserting foreign DNA into a vector.

  • Methods of Replication: Techniques (e.g., bacterial transformation, viral infection, PCR) to enable the replication of the recombinant DNA within a host cell or in vitro.

  • Methods to Identify/Select: Techniques to confirm the successful uptake and presence of the manipulated, recombinant DNA in the host cells, often involving selectable markers like antibiotic resistance genes or reporter genes.

Step 1 – Choose a Vector

• What is a Vector?: A molecular vehicle, typically a DNA molecule, used to transfer genetic material (the "insert" or "foreign DNA") into a host cell, where it can be replicated and/or expressed. The choice of vector is critical and depends on the specific goals of the cloning experiment, such as the size of the DNA insert, the desired host organism, and whether gene expression is required.

Plasmids as Vectors

• What is a Plasmid?:

  • Plasmids are small, circular (though some can be linear), extra-chromosomal DNA molecules found naturally in bacteria and some eukaryotic organisms (e.g., yeasts). They exist independently of the host's chromosomal DNA and replicate autonomously. They are also known as episomal DNA when they can integrate into the host chromosome, but typically refer to non-integrated, self-replicating elements.

  • Used in molecular biology to store and amplify foreign DNA and can express novel gene sequences, leading to the production of new proteins that may confer beneficial traits to the host cells, e.g., antibiotic resistance or metabolic enzymes.

  • Structure: Plasmids are typically double-stranded DNA, predominantly circular, but linear forms also exist. Rarely, they can be RNA. Bacterial plasmids are the most common source and are often engineered for various biotechnological applications.

Advantages of Plasmid Proteins to Hosts

• Examples of Beneficial Plasmid Encoded Proteins: Plasmids often carry genes that provide a survival advantage to their bacterial hosts. These include:

  • Heavy Metal Resistance: Genes encoding proteins that detoxify or efflux heavy metals (e.g., lead, mercury, arsenic) from the cell, providing a survival advantage in environments contaminated with these toxic substances. This can involve enzymes that modify the metal or membrane transport proteins.

  • Antibiotic Resistance: Genes that encode enzymes or proteins conferring resistance to various antibiotics. This allows bacteria to survive and proliferate in environments where antibiotics are present, enhancing their survivability in competitive ecological niches or clinical settings.

  • Fertility Factors: Plasmids like the F-plasmid carry genes (e.g., tra genes) that enable the bacterium to transfer copies of the plasmid DNA to other bacterial cells through a process called conjugation, a form of horizontal gene transfer. This rapidly spreads beneficial traits within a bacterial population.

  • Novel Protein Encoding: Enables bacteria to manufacture unique proteins, such as toxins, enzymes for degrading unusual substrates, or adhesion proteins, which can enhance virulence or metabolic versatility.

F-plasmid Genes

• Genes from F-plasmids (Fertility plasmids) orchestrate bacterial conjugation. These genes cause the synthesis of sex pili proteins, which are filamentous appendages on the bacterial surface. A pilus extends from a donor cell (F$^+$) and attaches to a recipient cell (F$^-$), establishing a stable cell-to-cell contact.

• Mobilization proteins (encoded by the F-plasmid) facilitate the processing and transfer of a single strand of the F-plasmid DNA through this cytoplasmic link (conjugation bridge) into the recipient cell. Both the donor and recipient cells then synthesize the complementary strand, resulting in both cells possessing a complete double-stranded F-plasmid. This converts the F$^-$ recipient cell into an F$^+$ donor cell.

• Some plasmid proteins can also facilitate the integration of plasmid DNA (or parts of it) into the new cell chromosomes (Hfr transfer).

Antibiotic Resistance Encoded by Plasmids

• Antibiotic | Target Protein/Enzyme | Effect | Resistance Mechanism

• Penicillin | Transpeptidase (cell wall synthesis) | Prevents cell wall linking, leading to osmotic lysis | Produces beta-lactamase, which hydrolyzes and destroys the beta-lactam ring structure.

• Ampicillin | Transpeptidase (cell wall synthesis) | Prevents cell wall linking | Inactivation of the drug by plasmid-encoded beta-lactamases.

• Tetracycline | 16S rRNA (ribosome A-site) | Inhibits binding of aminoacyl-tRNA, blocking protein synthesis | Membrane pump extrudes drug from the cell.

• Chloramphenicol | 23S rRNA (large ribosomal subunit) | Inhibits peptidyl transferase activity, blocking protein synthesis | Acetylation inactivates drug.

• Neomycin & Kanamycin | Ribosome – small subunit (30S subunit) | Causes mRNA misreading or blocks initiator tRNA binding; faulty protein synthesis | Modifying enzymes (e.g., phosphoryltransferases, adenylyltransferases) inactivate antibiotic; membrane pump extrudes drug from the cell.

• Trimethoprim | Dihydrofolate reductase (enzyme) | Inhibits dTTP and purine synthesis, blocking DNA synthesis | Produces an alternative, drug-resistant dihydrofolate reductase enzyme.

Plasmid Characteristics

• Plasmid Size and Copy Number: Plasmids vary significantly in size, ranging from a few kilobase pairs (kbp) to over a megabase pair (Mbp). The copy number refers to the average number of plasmid molecules present per host cell.

• Table of notable plasmids detailing host, size (kbp), structure, and copy number.

• Plasmid size and copy number typically exhibit a varied inverse relationship related to plasmid DNA replication control mechanisms. Smaller plasmids often have a high copy number because their replication is less tightly regulated (relaxed control), allowing many copies to accumulate per cell. Conversely, larger plasmids are usually present in a low copy number (stringent control) because their replication is more tightly coupled to chromosomal replication, maintaining a more stable ratio with the host chromosome. High copy number plasmids are generally preferred for gene cloning and protein expression experiments to maximize DNA yield or protein production.

Major Applications of Vectors

• Amplification of DNA: Vectors allow for the efficient replication of an inserted DNA fragment within a host cell, thus producing many identical copies of the DNA. This is fundamental for DNA sequencing, gene expression studies, and genetic engineering.

• Expression of recombinant proteins: By placing a gene of interest under the control of a suitable promoter in an expression vector, the vector can be used to direct the synthesis of the corresponding protein in a host cell (e.g., bacteria, yeast, mammalian cells). This is widely used in biotechnology for producing therapeutic proteins (e.g., insulin, growth hormones) or industrial enzymes.

• Construction of DNA libraries:

  • Genomic DNA libraries: A collection of recombinant vectors, each containing a different fragment of the entire genome of an organism. These libraries represent the complete genetic makeup of an organism and are used for gene discovery, genome mapping, and sequencing projects.

  • Complementary DNA (cDNA) libraries: A collection of recombinant vectors containing cDNA fragments, which are synthesized from mRNA using reverse transcriptase. These libraries represent only the genes that are actively expressed in a particular cell type or under specific conditions, making them useful for studying gene expression patterns and isolating protein-coding sequences.

• Gene therapy applications: Viral vectors (e.g., retroviruses, adenoviruses, AAVs) are engineered to deliver therapeutic genes into human cells to correct genetic defects or introduce new functions to treat various diseases like cystic fibrosis, severe combined immunodeficiency (SCID), and certain cancers.

• Development of improved recombinant vaccines and therapeutic peptides (e.g., Hepatitis B virus vaccine, insulin).

• Introduction of DNA into both prokaryotic and eukaryotic cells.

Features of a Vector

• Most commonly used cloning vectors (like plasmids) share several essential features that make them suitable for manipulating DNA:

  1. Origin of Replication (Ori): This is a specific DNA sequence that acts as a starting point for DNA replication, allowing the vector to be replicated independently within the host cell. The Ori determines the copy number of the plasmid.

  2. Multiple Cloning Site (MCS) or Polylinker: A short DNA segment containing several unique restriction enzyme recognition sites clustered together. This allows flexible insertion of foreign DNA fragments using different restriction enzymes without disrupting essential vector functions.

  3. Selectable Marker Gene: A gene that confers a selectable phenotype to host cells containing the vector (e.g., antibiotic resistance gene like ampicillin resistance, or a gene for prototrophy). This allows for the easy identification and selection of transformed cells from non-transformed cells.

  4. Reporter Gene (Optional but common): A gene that allows for visual screening of successful DNA insertion into the MCS. A common example is the lacZ gene, which, when interrupted by an insert, prevents the production of beta-galactosidase, leading to "white" colonies on X-gal-containing media (blue-white screening).

  5. Small Size: Smaller vectors are generally easier to handle, transform efficiently, and have higher copy numbers.

  6. High Copy Number (for amplification): For applications requiring large amounts of DNA, vectors with a high copy number are advantageous.

  7. Stable and Easy to Isolate: The vector DNA should be stable within the host and easily extracted in good yield and purity.

  8. Promoter (in expression vectors): For vectors designed for gene expression, a strong, regulatable promoter sequence is required upstream of the MCS to drive the transcription of the inserted gene into mRNA.

Types of Vectors

• Categories of Vectors: The choice of vector depends on the size of the DNA insert, the host organism, and the desired application.

  • Bacterial plasmids: Small, circular DNA molecules (e.g., pUC, pGEM, pBluescript series). Ideal for cloning small DNA fragments (up to ~$10$ kb) and for protein expression in bacteria due to their independent replication and selectable markers.

  • Bacteriophages (Phage vectors): Viruses that infect bacteria (e.g., M13, lambda ($ext{λ}$) phage).

    • M13 phage: Single-stranded DNA phage, useful for producing single-stranded DNA for sequencing or site-directed mutagenesis (cloning capacity ~$1-4$ kb).

    • Lambda ($ext{λ}$) phage: Double-stranded DNA phage, can accommodate larger DNA inserts (up to ~$25$ kb) and are efficient for constructing genomic libraries due to their high transformation efficiency.

  • Hybrids (e.g., Cosmids): These are plasmid-phage hybrid vectors that combine features of both. They are plasmids containing a bacteriophage $cos$ site (cohesive end site) that allows packaging into phage particles. They can carry larger DNA inserts (up to ~$45$ kb) than typical plasmids and are useful for cloning larger genes or gene clusters.

  • Artificial chromosomes: Engineered constructs designed to clone very large DNA fragments.

    • Bacterial Artificial Chromosomes (BACs): Derived from the F-plasmid of E. coli, capacity up to ~$300$ kb. They are low copy number vectors, important for minimizing recombination events with repetitive sequences.

    • Yeast Artificial Chromosomes (YACs): Can clone fragments up to ~$2$ Mb (2000 kb) in yeast. YACs possess centromeres, telomeres, and origins of replication, functioning like eukaryotic chromosomes.

  • Eukaryotic viruses: Used as vectors for gene delivery into eukaryotic cells (e.g., adenoviruses, retroviruses, adeno-associated viruses (AAVs)). These are essential for gene therapy and vaccine development.

  • Transposable elements (e.g., P elements): DNA sequences that can move (transpose) from one location in the genome to another. Used as vectors, particularly in model organisms like Drosophila (P elements), for insertional mutagenesis or gene delivery.

  • Considerations: Each type has specific advantages and disadvantages concerning insert size capacity, ease of manipulation, host range, and stable integration vs. transient expression.

Choosing a Vector: Considerations

• Important factors when selecting a vector are crucial for the success of a cloning or expression experiment. These include the application, host organism, insert capacity, complexity/ease of manipulation, and the desired copy number.

Choosing a Vector by Application

• High Copy Plasmids:

  • Make large quantities of plasmid DNA (e.g., pUC18/19, pGEM). They are ideal for cloning small-to-medium sized DNA fragments where high yield is beneficial.

• Single-stranded Phage Vectors:

  • Sequence or mutate DNA (e.g., M13). They are useful for producing single-stranded DNA for sequencing or site-directed mutagenesis.

• Protein Expression:

  • Utilize suitable powerful and regulatable promoters (e.g., T7 promoter in pET, pT7.7) to drive high levels of transcription and protein synthesis for the recombinant protein in a host cell. These often include tags for easier purification.

Points in Host Selection for Eukaryotic Proteins

• Bacterial Expression Limitations:

  • Bacteria do not properly phosphorylate eukaryotic proteins (e.g., p53) and lack the packaging system of eukaryotic cells (e.g., Endoplasmic Reticulum).

  • Bacteria are deficient in post-translational modifications like glycosylation, disulfide bond formation, and lipidation, often leading to misfolded or non-functional proteins. Differences in codon usage frequency also affect translation efficiency.

• Therefore, eukaryotic cell culture systems (e.g., yeast, insect cells, mammalian cells) are generally recommended for expressing functional, modified eukaryotic proteins as they provide the necessary machinery for proper synthesis, folding, and post-translational processing.

Manipulating Plasmids with Restriction Endonucleases

• Bacterial Immune System Role: Restriction endonucleases (RE) function as a defense mechanism against invading foreign DNA by recognizing specific sequences and cutting them. Bacteria protect their own DNA by methylating these sequences.

• Known REs: Over 4000 known, with hundreds commercially available.

• Types of Endonucleases:

  • Type I REs: Cut at random sites, far (up to $1000$ bp) from their recognition sequences, requiring ATP and SAM. Generally not useful for molecular biology due to imprecise cutting.

  • Type II REs: Cut precisely within or very close to specific palindromic recognition sequences, usually $4-8$ bp long. Most useful for molecular biology applications as they produce predictable DNA fragments and require only Mg$^{ ext{2+}}$.

Type II REs Characteristics

• Cutting Precision: Type II REs cut precisely at predetermined target sequences, cleaving phosphodiester bonds on both strands of double-stranded DNA. They do not act on single-stranded DNA or RNA.

• Naming Convention: Type II REs receive names based on the organism from which they were isolated (e.g., EcoRI from Escherichia coli, R strain I).

Different Types of REs Yield Varied Ends

• Examples of Specific Restriction Enzymes: The nature of the DNA ends produced is critical for subsequent ligation reactions:

  • EcoRI: Cuts sequence $5'$-G^AATTC-$3'$, producing cohesive $5'$ overhangs. These complementary sticky ends greatly increase ligation efficiency.

  • FspI: Produces blunt ends when cutting $5'$-TGC^GCA-$3'$. Blunt-ended fragments are less efficiently ligated.

  • KpnI: Produces cohesive $3'$ overhangs when cutting $5'$-GGTAC^C-$3'$.

DNA Ligases and Their Functions

• Repair Mechanism: DNA ligases are responsible for repairing discontinuities (nicks) in the phosphodiester backbone of DNA strands, crucial in DNA repair, replication (joining Okazaki fragments), and recombination.

• Function of Ligase: DNA ligase forms a single phosphodiester bond between the $3'$-hydroxyl group of one nucleotide and the $5'$-phosphate group of an adjacent nucleotide in the same DNA strand; this seals a nick in double-stranded DNA.

• ATP Hydrolysis Requirement: ATP must be hydrolyzed for T4 DNA ligase activity, as AMP covalently binds to the ligase (specifically, lysine-34 in T4 DNA ligase). This activates the ligase, allowing it to transfer AMP to the $5'$-phosphate of one DNA strand, which is then attacked by the $3'$-hydroxyl of the adjacent strand to form the phosphodiester bond.

Ligation Considerations

• Choosing appropriate restriction enzymes to clone specific DNA fragments into vectors is crucial, with ligase joining any compatible cohesive ends (or blunt ends).

• Re-Circularization Issue: Linearized plasmids, once cut by a single RE, have a strong tendency to ligate to themselves (re-circularization), which is a competing reaction against ligation to the respective insert DNA. This leads to a high background of non-recombinant plasmids.

• Prevention of Re-Circularization: Phosphatases (e.g., Calf Intestinal Alkaline Phosphatase, CIP) can prevent these undesirable re-circularization events by removing the $5'$-phosphate groups from the linearized vector, ensuring it cannot self-ligate. The insert DNA must retain its $5'$-phosphates for successful ligation to the vector.

Cloning Techniques Using PCR

• Cloning through PCR: This section involves using primers to amplify specific portions of the genome through PCR methods, often incorporating restriction enzyme sites at their ends. The PCR product and vector are then digested and ligated.

• Directional Cloning: Ensures that the PCR product is inserted in a specific orientation, often with the help of two different restriction sites incorporated into the primers and vector (e.g., HindIII, NotI) to create non-compatible cohesive ends. This is vital for proper gene expression.

• For expression vectors, the T7 promoter is utilized for upstream transcription initiation by T7 RNA polymerase for high-level downstream gene expression.

Identifying and Purifying Recombinant Clones

• Key steps include manipulating bacterial cells to take in and replicate vectors and robustly confirming the incorporation of the desired plasmid DNA with the correct insert.

• Distinction between Cloned Sequences: Differentiation between cells with or without plasmid, and between original (empty) and recombinant vectors with the insert, through specific selection and screening processes, typically involving antibiotic resistance markers and reporter genes.

Transforming Bacteria

• Transformation Methods: Utilized to induce competent bacteria (e.g., JM109) to take in plasmids by creating a transient permeable state in their cell walls.

  • Heat Shock: Incubation with cold divalent cations ($ext{Ca}^{ ext{2+}}$ or $ext{Rb}^{ ext{2+}}$) neutralizes DNA and membrane charges. A brief heat shock ($42^ ext{o} ext{C}$ for $30-90$ seconds) creates temporary pores, allowing plasmid DNA to enter.

  • Electroporation: A high-voltage electrical pulse creates transient pores, offering higher transformation efficiency, especially for larger plasmids.

• Selection of Transformants: Select transformants using antibiotic resistance markers by incubating cells on nutrient agar containing antibiotics. An outgrowth period allows expression of the resistance gene.

Transformants and Clonal Selection

• Growth of Transformants: All bacteria that successfully uptake a replicating plasmid are termed transformants and can grow in antibiotic presence due to antibiotic resistance gene expression.

• Selection Principle: Non-transformants lack plasmids and thus cannot express antibiotic resistance, failing to grow on selective media. This leads to clonal populations of cells containing the desired plasmid.

Screening and Selection Processes

• The Importance of Screening: After ligation and transformation, verification of the presence and integrity of the recombinant DNA is crucial for successful cloning operations and to identify true recombinant clones.

• Pathways to Identification: Methods to distinguish recombinant plasmids with the insert from original (empty) forms:

  • Blue-White Screening: Using a lacZ reporter gene; disruption by an insert leads to white colonies, while functional lacZ gives blue colonies.

  • Colony PCR: Amplifying DNA directly from colonies to check for insert presence and size.

  • Restriction Digest Analysis: Isolating plasmid DNA and digesting it to confirm insert presence, size, and orientation via gel electrophoresis.

  • DNA Sequencing: The definitive method to confirm the exact sequence of the insert and its orientation within the vector.

Alternative Cloning Approaches

• Recombineering: Involves bacteriophage-derived recombinase enzymes (e.g., Red/ET system) for highly efficient direct insertion of PCR DNA into target sites on plasmids or chromosomes in vivo using homologous recombination. This bypasses restriction enzymes and ligases.

Vector Characteristics and Cloning Capacity

• Vector Structures and Capacity: Table of various vectors indicating structure, host, and insert size. The choice of vector depends on the size of the DNA fragment to be cloned and the stability required.

Importance of BACs and YACs in Large Fragment Cloning

• BACs (Bacterial Artificial Chromosomes): Derived from the F-plasmid of E. coli, possess a low copy number (1-2 per cell) for high stability. Capable of cloning fragments from $50$ kb up to ~$300$ kb, they are extensively used for genomic libraries and physical mapping.

• YACs (Yeast Artificial Chromosomes): Enable cloning of very large DNA fragments (up to ~$2$ Mb) in Saccharomyces cerevisiae. YACs function like eukaryotic chromosomes with centromeres, telomeres, and origins of replication, valuable for large gene clusters and whole-genome studies.

Viral Vectors in Gene Delivery

• Virus Types for Cloning:

  • Lambda phage ($ext{λ}$): Contains genes necessary for structure and replication. Lambda cloning vectors optimize space for foreign DNA (typically $2-25$ kb) by deleting non-essential gene clusters and rely on specific cohesive ends ($cos$ sites) for efficient packaging into phage heads in vitro.

Gene Therapy Approaches with Viral Vectors

• Applications of Gene Therapy: Use of engineered viral vectors to introduce corrected copies of mutated genes or new genes to treat diseases, such as cystic fibrosis, hemophilia, certain cancers, and inherited immune deficiencies.

• Retrovirus Usage: Retroviral vectors allow for stable genome integration of the therapeutic gene into the host cell's DNA, leading to long-term expression. This makes them suitable for treating chronic conditions like severe combined immunodeficiency (SCID) and potentially HIV/AIDS.

Concerns with Various Vector Types

• Insertional Mutagenesis Risk: A significant concern, especially with retroviral vectors, is that unpredictable integration sites can disrupt host genes (e.g., proto-oncogenes or tumor suppressor genes), potentially leading to oncogenesis (cancer development).

Overview of Various Viral Vectors

• Adenovirus: Historically associated with common cold; serves as a vector but typically remains episomal (does not insert into host DNA), making expression transient and reducing integration risk.

• Adeno-associated Virus (AAV): Known for non-pathogenic characteristics and, in its wild-type form, site-specific integration into chromosome 19. Recombinant AAVs are favored for their safety, broad tropism, and ability to transduce both dividing and non-dividing cells, providing stable, long-term expression (often episomally).

Global Gene Therapy Developments

• Glybera (alipogene tiparvovec): Notably recognized as the first approved gene therapy in Europe (2012), aimed at treating lipoprotein lipase deficiency. It used an AAV vector to deliver a functional gene. However, it faced scrutiny regarding efficacy, high cost (€$1.1$ million per treatment), and limited market viability, leading to its commercial withdrawal in 2017.

Viral Vector Vaccines

• Mechanisms of Action: Recombinant viruses function as vaccine delivery systems by inserting genes encoding specific antigens (e.g., SARS-CoV-2 spike protein) into a harmless viral vector. The vector infects host cells, which then express and present these antigens to the immune system, generating a robust immune response (humoral and cellular) without causing disease.

• Next-Generation Vaccine Platforms for COVID-19 employing adenoviral vectors (e.g., AstraZeneca, Johnson & Johnson) have shown significant efficacy in creating robust immune responses and protection.

Summary of Gene Therapy and Molecular Cloning

• Transformative Potential: Highlight the ability of gene therapy and molecular cloning processes to provide fundamental tools for biological research and curative options for various genetic disorders. Their ongoing development continues to revolutionize medicine, contributing to novel diagnostics, advanced therapeutics, and next-generation vaccines.