Recombinant DNA Technology

Recombinant DNA Technology

  • Introduction to Recombinant DNA Technology: Processes for genetic engineering by altering nucleotide sequences in genomes.

Learning Objectives

  • Understanding the steps involved in cloning:
    • What is necessary at each stage?
    • How do these components function?

Key Terminology

  • Genetic Engineering: Deliberate modification of an organism's genetic information.
  • Recombinant DNA Technology: Procedures used to perform genetic engineering.
  • Cloning: Generation of a large number of genetically identical DNA molecules.
  • Biotechnology: The use of living organisms to create useful products.

Gene Expression in E. Coli

  • To express a gene of interest, specific techniques and tools are followed.

Important Tools in Recombinant DNA Technology

  • PCR (Polymerase Chain Reaction): Amplifies specific DNA fragments.
  • Gel Electrophoresis: Separates DNA, RNA, and protein by size.
  • Restriction Enzymes and Ligase:
    • Restriction Enzymes: Cut DNA at specific sequences.
    • Ligase: Joins DNA fragments.
  • Cloning: Involves replicating DNA within a plasmid.

The Polymerase Chain Reaction (PCR)

  • Enables gene amplification, which is the rapid synthesis of many DNA copies from a mixture.
  • Components of PCR:
    • Primers, target DNA, thermostable DNA polymerase (e.g., Taq), and dNTPs.
  • Thermocycler: Instrument used in the reactions.
    • Phases of PCR:
    1. Denaturation (~100°C): DNA strands are separated.
    2. Annealing (55-60ºC): Primers bond to target DNA.
    3. Extension (~70°C): DNA polymerase adds nucleotides to the DNA strand.
  • Cycles of PCR result in exponential amplification of DNA, doubling the amount each cycle.

Uses of PCR

  • Simplifies gene cloning.
  • Produces DNA fragments for sequencing.
  • Diagnoses diseases (e.g. AIDS, tuberculosis).

Gel Electrophoresis

  • Used for separating DNA based on charge and weight.
    • DNA migrates towards the positive end due to its acidity.
  • Migration rate inversely proportional to log of molecular weight.

Southern Blotting Technique

  • Developed by Edwin Southern in 1975; a 3-step process:
    1. Separate DNA molecules.
    2. Transfer separated DNA to a membrane.
    3. Hybridize with a labeled DNA probe.

Northern and Western Blots

  • Northern Blot: Similar to Southern but works with RNA using agarose gel.
  • Western Blot: Used for protein analysis on polyacrylamide gel.

Restriction Enzymes

  • Recognize specific DNA sequences and cleave at those sites.
  • Produce either sticky or blunt ends.
  • Hundreds available commercially for cloning applications.

Genetic Cloning and cDNA Synthesis

  • Historical context of first recombinant DNA (Jackson, Symons, Berg, 1972).
  • Plasmids as vectors for foreign DNA insertion.

Ligation of DNA Fragments

  • DNA Ligase: Joins the phosphate backbone of DNA fragments.
    1. Molecules collide.
    2. 3'-OH connects to 5'-phosphate, forming a covalent bond.
  • Requires ATP for ligase activity.

Plasmids and Cloning Requirements

  • Plasmids: Easily purified; can autonomously replicate.
  • Vector requirements include:
    • An origin of replication.
    • A selectable marker.
    • A multicloning site (polylinker) for inserting genes.

Artificial Chromosomes

  • Used to clone large DNA fragments:
    • Bacterial Artificial Chromosomes (BACs): Up to 300 kb.
    • Yeast Artificial Chromosomes (YACs): For larger fragments up to 1,000 kb.

Cosmids

  • Engineered vectors containing features from both plasmids and phages.
  • Capable of containing inserts larger than regular plasmids.

Sequencing Techniques

  • Sanger Method: Most common; uses ddNTPs to terminate DNA strands at specific bases.
  • Next-Generation Sequencing (NGS): Faster and cheaper method involving massively parallel sequencing; does not require cloning fragments into vectors.
  • Automated Sanger Sequencing: Uses fluorescent dyes for sequencing, allowing rapid results.

Metagenomics and Transcriptomics

  • Metagenomics: Analyzing genetic material from environmental samples without culturing.
  • Transcriptomics: Studies mRNA production to analyze gene expression.

RNA Sequencing (RNA-Seq) Advantages and Implementation

  • More sensitive and accurate than microarrays; no need for probes reduces bias.
  • Measures mRNA levels to compare expression profiles or identify transcripts.

Problems with Microarrays

  • Limitations include inability to detect all mRNAs, potential noise from non-specific bindings, and limited dynamic range for quantification.

Data Acquisition in RNA-Seq

  • Involves library preparation, sequencing, and computational analysis to quantify gene expression and construct gene models.