(589) 17B_1 Tools for DNA Technology

Introduction to Biotechnology and Genetic Engineering

  • Focus on combining knowledge of DNA, RNA, and protein function to explore biotechnology and genetic engineering.

  • Overview of key tools to sequence, study, and manipulate DNA, including:

    • DNA sequencing

    • Gene cloning

    • PCR amplification

  • Objectives include expressing genes, analyzing expression, and gene editing.

DNA Technologies

  • Key Techniques:

    • DNA sequencing

    • DNA cloning

    • Gene expression

    • Gene editing

  • Central to these techniques is the ability of nucleic acids to bind to complementary strands (nucleic acid hybridization) - RNA or DNA base pairs with complementary sequences.

  • Definitions:

    • Genetic Engineering: Direct manipulation of genes for practical purposes.

    • Biotechnology: Using organisms or components to produce useful products (e.g., brewing beer using yeast).

DNA Sequencing Methods

  • Sanger Sequencing:

    • Developed by Fred Sanger; first automated DNA sequencing method.

    • Uses dideoxy method where sequencing involves stopping synthesis at specific bases.

    • Generates a gel with bands indicating the nucleotide positions.

  • Next-Generation Sequencing Technologies:

    • Allows sequencing of large fragments (~300 nucleotides) in parallel.

    • Sequences ~2 billion nucleotides in 24 hours.

    • Sequencing by Synthesis:

      • DNA fragments attached to beads, mixed with DNA polymerases and primers to read the strand via fluorescence.

Advanced Sequencing Techniques

  • Nanopore technology allows for direct sequencing through changes in electrical current as bases pass through pores.

  • Significant reduction in time and cost for sequencing genomes (e.g., human genome project took 13 years and $1 billion in 2003).

Gene Cloning

  • Importance of Cloning:

    • Create multiple copies of a gene for research or protein production.

    • Genes are inserted into plasmids (circular DNA molecules) to work with them in bacteria.

  • Methodology:

    • Use restriction enzymes to cut plasmids and target DNA, creating matching sticky ends for base pairing.

    • DNA ligase joins these fragments to produce recombinant DNA.

  • Types of Plasmids:

    • Cloning vectors: for copying genes.

    • Expression vectors: for producing proteins.

Properties of Plasmid Vectors

  • Plasmids carry important features:

    • Origin of replication

    • Antibiotic resistance genes for selection (e.g., ampicillin resistance)

    • Multiple cloning site with restriction enzyme recognition sequences.

Restriction Enzymes

  • Definition: Enzymes that specifically cut DNA at recognition sites, producing restriction fragments.

  • Recognition sites: Often palindromic sequences; they generate sticky ends for easy recombination.

  • Example: EcoRI recognizes GAATTC and cuts between A and G, creating sticky ends with AATT.

  • Applications:

    • Useful in genetic engineering to join DNA from different sources efficiently.

DNA Ligase and Assembly of Recombinant DNA

  • DNA ligase seals gaps in the DNA backbone after sticking gene fragments together using sticky ends.

  • The result is a recombinant plasmid containing the inserted gene for further study or protein production.