Microbial DNA Technologies - BIO 2280 Lecture 18

Microbial DNA Technologies - BIO 2280 Lecture 18

Recent Advances in Genomic Sequencing

  • Most Complete Human Genome: An international consortium has sequenced the most comprehensive version of the human genome over 20 years since the first draft.

    • There are now an additional 200 million bases that fill previously missing gaps.

    • Key Features Included:

    • Telomeres: Protective end-caps of chromosomes.

    • Centromeres: Central dense knobs that orchestrate replication.

    • Significant Update: The Y chromosome has been sequenced, providing insights into previously invisible regions of the genome.

    • Quote: "It really gives us some insight into regions of the genome that have been invisible" - Deanna Church, genomicist.

    • Source: Science, April 2022.

Key Concepts in Microbial DNA Technology

  • Sections Covered:

    • 17.1 Key Discoveries Leading to Recombinant DNA Technology

    • 17.2 Polymerase Chain Reaction (PCR) Amplifies Targeted DNA

    • 17.3 Genomic Libraries: Cloning Genomes in Pieces

    • 17.4 Expressing Foreign Genes in Host Cells

17.1 Key Discoveries Led to the Development of Recombinant DNA Technology

  • Terminology:

    • Genetic Engineering: The modification of the genetic code of living organisms, including crop improvement and animal breeding.

    • Recombinant DNA Technology: The process involves creating DNA molecules with segments from different organisms.

    • DNA Cloning: The use of enzymes and bacterial cells to modify and amplify DNA.

    • Biotechnology: The application of organisms in the production of useful products.

  • Key Discoveries contributing to DNA Cloning Technology:

    • Restriction Enzymes

    • Genetic Cloning and cDNA Synthesis

    • Cloning Vectors

    • Gel Electrophoresis

Restriction Enzymes

  • Importance: Recognize and bind to specific sequences in DNA known as recognition sites.

    • Cleave DNA at the recognition site or at a specified distance from it.

    • Can produce sticky ends or blunt ends in the DNA target.

    • Analysis of digests typically performed by gel electrophoresis.

    • Named for the bacterium from which they originate, with hundreds commercially available.

Common Restriction Endonucleases (Table 17.1)

  • Alul

    • Microbial Source: Arthrobacter luteus

    • Recognition Sequence: 5' AGCT 3' / 3' TCGA 5'

    • Ends Produced: Sticky ends

  • BamHI

    • Microbial Source: Bacillus amyloliquefaciens H

    • Recognition Sequence: 5' GGATCC 3' / 3' CCTAGG 5'

    • Ends Produced: Sticky ends

  • EcoRI

    • Microbial Source: Escherichia coli

    • Recognition Sequence: 5' GAATTC 3' / 3' CTTAAG 5'

    • Ends Produced: Sticky ends

  • HindIII

    • Microbial Source: Haemophilus influenzae d

    • Recognition Sequence: 5' AAGCTT 3' / 3' TTCGA 5'

    • Ends Produced: Sticky ends

  • NotI

    • Microbial Source: Nocardia otitidis-caviarum

    • Recognition Sequence: 5' GCGGCCGC 3'

    • Ends Produced: Sticky ends

  • SalI

    • Microbial Source: Streptomyces albus

    • Recognition Sequence: 5' GTCGAC 3'

    • Ends Produced: Sticky ends

  • Note: Arrows indicate where cleavage occurs on each strand.

Genetic Cloning and cDNA Synthesis

  • Pioneered by Jackson, Symons, and Berg in 1972, who generated the first recombinant DNA molecules.

    • Process involves recombining foreign DNA into a plasmid vector.

    • Function of Plasmid Vectors: These vectors replicate and maintain foreign DNA fragments.

cDNA Synthesis and Reverse Transcriptase

  • Reverse Transcriptase: An enzyme that synthesizes double-stranded DNA (cDNA) from RNA templates (mRNA).

    • Independently discovered by Temin and Baltimore in 1970.

Gel Electrophoresis

  • Used to separate DNA molecules based on their charge and molecular weight.

    • Materials: Agarose or acrylamide gels can be used to separate DNA fragments.

    • Mechanism: DNA is negatively charged; it migrates from the negative to the positive end of the gel.

    • Each fragment's migration rate is inversely proportional to the log of its molecular weight.

Cloning Vectors

  • Function: DNA elements that replicate and carry additional genetic cargo to facilitate cloning.

    • Different types include:

    • Plasmids: Small, circular DNA molecules that replicate autonomously.

    • Bacteriophages: Viruses that infect bacteria, genetically modified for DNA transfer.

    • Viruses: Used in genetic engineering; commonly retroviruses, lentiviruses, and adenoviruses in animal cell engineering.

    • Cosmids: Hybrid vectors that combine features of plasmids and bacteriophages.

    • Artificial Chromosomes: Used for cloning large DNA fragments, including bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs).

Requirements for Cloning Vectors

  • Essential Features:

    • Origin of replication: Allows the vector to be replicated within the host cell.

    • Selectable marker: A gene that enables the identification of transformed cells.

    • Multi-cloning site (MCS): A segment allowing the insertion of foreign DNA for cloning purposes.

17.2 The Polymerase Chain Reaction (PCR) Amplifies Target DNA

  • Purpose: PCR enables the amplification of a specific DNA fragment from a complex mixture of DNA and cellular components.

    • Uses Oligonucleotides Primers: Single-stranded DNA molecules, typically 15 to 30 nucleotides long.

    • Only DNA with known (or nearly known) sequences can be amplified with PCR.

PCR Reaction Mix Composition

  • Components of a typical PCR reaction mix include:

    • Template DNA: The DNA to be amplified.

    • Two Primers: Complementary to the target sequence.

    • Thermostable DNA Polymerase: Such as Taq polymerase, which withstands high temperatures.

    • Four Deoxyribonucleotide Triphosphates (dNTPs): Necessary for DNA synthesis.

  • Thermocycler: Device used to conduct the PCR reaction, cycling through different temperature stages for denaturation, annealing, and extension.

The PCR Technique

  • Cycle 1:

    • Denaturation: Heating to separate DNA strands.

    • Cycles Result: Each cycle doubles the number of target DNA molecules.

    • Cycle 2 yields 4 molecules, and Cycle 3 yields 8 molecules.

  • Final Result: After several cycles, the majority of fragments will consist only of the region flanked by primer sites.

Applications of PCR

  • Gene Isolation: From complex samples like soil, water, and blood.

  • Diagnosis: Essential for rapid and specific tests for infections like Chlamydia, hepatitis, and human papillomavirus.

  • Forensic Science: Crucial in DNA fingerprinting technology used in criminal investigations.

17.3 Genomic Libraries: Cloning Genomes in Pieces

  • Genomic Library Construction: Used for cloning genes from chromosomes that have not been sequenced.

    • Constructed by cleaving the genome into fragments and cloning them into vectors.

    • Libraries can be screened for genes of interest.

    • cDNA Library: Constructed for DNA fragments without intron sequences.

Construction and Screening Steps

  • Sequencing Process:

    • Genomic DNA is cleaved using restriction enzymes.

    • Cloned fragments are transformed into host cells (referred to as transformants).

    • Specific media used for screening ensure only colonies with the gene of interest can grow.

17.4 Expressing Foreign Genes in Host Cells

  • Heterologous Genes: Cloned genes in new host cells that require modifications for expression.

    • Recombinant Genes: Must have a promoter recognized by host RNA polymerase.

    • Differences exist in eukaryotic and bacterial systems (e.g., modifications required for mRNA).

  • Expression Vectors: Essential for overcoming problems with the expression of recombinant genes; they contain inducible promoters to drive high-level transcription.

Purification and Study of Recombinant Proteins

  • Protein Purification: The process of isolating the gene product, often utilizing techniques such as:

    • Polyhistidine Tagging (His-tagging): Attaching a polyhistidine sequence to facilitate purification via metal affinity chromatography.

    • Fluorescent Labeling: Coupling genes to fluorescent peptides for visualization during in vivo expression and regulation.

Steps for Construction and Purification of a Polyhistidine-Tagged Protein

  • Cloning: The gene encoding the protein is cloned into an expression vector containing histidine residues.

  • Transformation: Plasmid introduced into suitable host cells, like E. coli.

  • Culturing: Host cells are cultured to express many His-tagged protein molecules.

  • Purification Process:

    • Cells are lysed to extract soluble materials.

    • Soluble materials applied to a metal ion resin column.

    • Unbound proteins washed away.

    • His-tagged protein eluted from the column with imidazole.

Concept Check Questions

  • For PCR:

    • Define the function of each of the three steps in a PCR cycle.

    • Summarize the importance of PCR in biology.

  • For Genomic Libraries:

    • Explain why genomic and metagenomic libraries are useful.

  • For Recombinant Genes:

    • Discuss the utility of expression vectors in genetic engineering.