Gene cloning

Gene Cloning and Recombinant DNA Technology in Medicine

Prof. Bart Dzudzor

Recombinant DNA Technology

  • Involves techniques in manipulating DNA:
    • Molecular Cloning
    • Polymerase Chain Reaction (PCR)
    • Southern and Northern analysis
    • Production of proteins

Restriction Endonucleases

  • Their discovery revolutionized recombinant DNA technology by allowing the cut of DNA double helix at specific nucleotide sequences.
  • Purified from bacteria.
  • Characteristics:
    • Recognizes short sequences of DNA (usually 4-8 bp long) and cuts both strands.
    • Protection from cleavage in bacterial genomes occurs via methylation at A or C residues; foreign DNA is generally not methylated.
    • Recognition sequences are typically palindromic, meaning they read the same forward on one strand and backward on the opposite strand.
  • Types of cuts:
    • Staggered cuts (producing "sticky ends" or "cohesive")
    • Blunt cuts (producing "blunt ends")
  • Fragments from cleavage are termed restriction fragments.
  • Sticky ends can transiently base-pair, and these can be covalently joined by DNA ligase.

Applications of Restriction Enzymes

  • Used for:
    • Fractionating genomic DNA by size (Southern blotting)
    • Cloning specific sequences (generating libraries)
    • Mapping regions of DNA (restriction site mapping)
    • Isolating specific fragments of DNA (to construct labeled probes)

Gene Cloning Importance in Medicine

  • Reasons for gene cloning by medical researchers:
    • To define inherited genetic mutations that cause or predispose diseases.
    • To isolate functional (“normal”) genes for therapeutic molecule production.
    • To isolate genes containing somatic mutations (acquired, not inherited).
    • To gain an enhanced understanding of biochemical pathways (signal transduction, immunity, development, cell cycle).

Molecular Cloning

  • Definition: to study and manipulate a particular DNA sequence or gene, the process called cloning duplicates it into millions of identical copies.
  • Differentiation from organism cloning: Cloning organisms regenerates identical copies from a single cell.
  • Process involves:
    • Joining a specific DNA fragment to a replicating DNA molecule (vector), usually in E. coli.
    • Common vectors include plasmids and bacteriophages. Eukaryotic systems also use plasmids and viruses (retroviruses).

Features of Cloning Vectors

  • Essential features of all vectors:
    • Origin of replication
    • Multiple cloning sites (polylinker region)
    • Selectable marker (antibiotic resistance gene)
  • Expression vectors must also contain:
    • Promoter region

Cloning Procedure

  • General steps:
    1. Isolate desired DNA fragments from mixtures by cloning them into a plasmid vector.
    2. Isolate plasmid DNA from cells in colonies for characterization or further manipulation.

Uses of Cloning

  • Applications of cloning include:
    1. Isolating and characterizing specific genes or mRNA from complex mixtures, such as the genome or total cellular RNA.
    2. Generating probes for detecting homologous sequences, aiding in disease diagnosis.
    3. Expressing large quantities of proteins, which are useful for therapeutic purposes.
    4. Developing animal models with specific mutations for disease study.

DNA Libraries

  • Libraries consist of collections of DNA fragments inserted into vectors.
    • Genomic Libraries: Contain DNA from an organism's genome, including all elements such as coding regions, introns, regulatory elements, etc.
    • cDNA Libraries: Contain double-stranded DNA copies from mRNA, reflecting only expressed sequences.
  • cDNA synthesis uses reverse transcriptase to create a single strand, later converted to double-stranded DNA using DNA polymerase and cloned into vectors.

Constructing and Using Genomic Libraries

  • Contains all genetic information (including introns, exons, non-coding regions).
  • Reasons for constructing and screening genomic libraries include:
    1. Cloning whole genes (including introns and regulatory sequences) for gene expression studies.
    2. Cloning within chromosomal context to identify neighboring genes.
    3. Creating knockout models by replacing functional genes with non-functional copies.

Considerations for cDNA Libraries

  • Important points regarding cDNA libraries:
    • No upstream regulatory sequences, since these are not included in mRNA.
    • Source of mRNA is critical—no expression means no cDNA in the library.
    • Absence of introns allows prediction of protein sequences from cDNA.
    • Cannot determine chromosomal location or relationship to neighboring genes.

Southern Blot Technique

  • Technique to detect specific DNA sequences.
  • Process for Northern Blotting involves denaturation of total RNA using agents like formaldehyde, ensuring linear conformation for analysis.
  • Southern blot diagnostics include:
    • Detection of large insertions/deletions (50-100 bp) affecting hybridizing probe sizes.
    • Identifying gross gene amplification (normal diploid cells usually have 2 copies).
    • Detect genomic rearrangements caused by chromosomal translocations (e.g., BCR-Abl in chronic myeloid leukemia).
    • Single nucleotide mutations detected only if they alter restriction enzyme sites (as in hemophilia A and sickle cell disease).

Limitations of Southern Blots

  • Southern blot does not detect:
    • Gene expression status.
    • Point mutations (unless they affect restriction enzyme sites).
    • Small deletions.

Northern Blot Technique

  • Useful for detecting:
    • Expression of specific genes across different tissues (excluding housekeeping genes).
    • Size determinations of RNA transcripts, which could indicate deletions or insertions.
    • Relative expression levels in varied samples, linked to disease states.
  • Can identify deletions or insertions and splicing defects in genes.

Polymerase Chain Reaction (PCR)

  • Procedure amplifying specific DNA sequences without cloning into vectors—crucial in clinical and forensic contexts with limited DNA.
  • Utilizes short, chemically synthesized primers flanking the target region.
  • Employs thermostable DNA polymerase (from Thermus aquaticus) and involves:
    • Heating to melt template DNA, followed by primer annealing at lower temperatures.
    • Repeating amplification through polymerase and dNTP incorporation.
  • Generally cycled 20-40 times, with each cycle doubling the DNA amount; e.g., 20 cycles yield $2^{20} = 1,048,576 $ copies.

Applications of PCR

General uses include:

  • Detection of mutations (inherited or acquired) via PCR followed by DNA sequencing.
  • Identifying Minimal Residual Disease in leukemia using sequences near chromosomal breakpoints.
  • Gender determination in embryonic cells from in vitro fertilization when X-linked mutations are present.
  • In vitro mutagenesis testing specific mutations’ effects on gene function.
Additional applications include:
  • Gene cloning
  • Carrier screening
  • Clinical diagnosis/confirmation
  • Newborn screening
  • Presymptomatic diagnosis/predisposition screening
  • Transplant engraftment analysis post transplantation
  • Parentage/paternity testing
  • Forensic identification (matching suspect DNA to crime scene evidence)
  • Distinguishing monozygotic from dizygotic twins
  • Early detection of HIV
  • Addressing surgical specimen mix-ups
  • DNA fingerprinting
  • Prenatal diagnosis utilizing various sample types:
    • Amniocentesis
    • Chorionic villus sampling (CVS)
    • Embryonic blastomeres for preimplantation diagnosis
    • Fetal cells circulating in maternal blood

Types of DNA Polymorphism

  • RFLP (Restriction Fragment Length Polymorphism): Variations in restriction enzyme recognition sites; detectable via Southern blotting or PCR.
  • VNTR (Variable Number of Tandem Repeats): Repeated nucleotide sequences differing between individuals, also known as minisatellites.
  • STR (Short Tandem Repeats): Short specific nucleotide sequences (2-8 nucleotides) differing in repeat number; also referred to as microsatellites.
  • Relationship between individuals can be studied as more related individuals will have similar STRs.
  • Detection of SSR variations is achievable through PCR and gel electrophoresis, resulting in product sizes reflecting repeat numbers.

Continuing DNA Polymorphism Discussion

  • SNP (Single Nucleotide Polymorphism): Nucleotide differences between individuals that do not coincide with restriction enzyme sites; studied by PCR and gel electrophoresis.
  • HLA Genes: Highly polymorphic genes on chromosome 6p, useful for identity studies, including DNA level and antibody recognition of protein variations.

SSR Inheritance Example

  • Example demonstrating inheritance of SSR:
    • Parent 1 (P1): (TCTA)10
    • Parent 2 (P2): (TCTA)11
    • Children inherit variable repeats, leading to differences in repeat numbers across generations.

Production of High Levels of Proteins from Cloned cDNA

  • Once desired cDNA is cloned, proteins can be produced in engineered E. coli cells.
  • Example: Granulocyte colony-stimulating factor is synthesized in expression vectors optimized for high-level protein production.

Conclusion

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