Recombinant DNA Technology

Overview

1. Cloning Genes: The Tool Kit
   • Enzymes
   • Vectors
   • Host Cells
2. Identifying specific clones by Screening
3. PCR Cloning without Host cells
4. Characterizing Clones
5. DNA Sequencing

Cloning DNA

1. Restriction enzymes to produce DNA fragments.
2. Combine with vector DNA to produce recombinant DNA molecules (host cell).
3. Transfer recombinant vectors into the host cell to clone the recombinant DNA molecules.

Restriction Enzymes

• They cut DNA into fragments
• They are made by bacteria as a defense against bacteriophages
• Recognizes a specific sequence (restriction site) and cuts the DNA into fragments.
• Cleaves both strands of DNA.

Recognition Sequences are often Palindromes

• Palindrome:
  • Symmetry exhibited by recognition sequences.

Making Recombinant DNA molecules

• Taking DNA from two different sources into a single molecule. (Recombinant DNA)
• Take DNA from each source.
• Cut each one with the same restriction enzyme.
• Mix.

DNA Cloning with Plasmids

• Plasmid:
  • Goal is to use a carrier molecules
  • They replicate independently from the chromosomes within bacterial cells.
  • Extrachromosomal double-stranded DNA.
• Plasmids used in DNA cloning:
  • Genetically modified bacterial plasmids engineered to contain:
    • A convenient amount of restriction sites.
    • A marker gene to select for presence in host cell.
• Procedure
  • Isolate DNA
  • Cut cloning site
  • Transfer recombinant vectors to host cell
  • Grow them on nutrient medium
  • Check colonies that contain the recombinant vector

Identifying Specific Clones

• Requires a tag called a probe to identify specific clones.
• Probe: a DNA (or RNA) molecule that has been labeled with radioactivity or a fluorescent dye.
• Uses molecular hybridization.

Molecular Hybridization

• What is it:
  • Probes are used as targets for lysing.
  • Reactions are carried out on a substrate and not a solution.
• How:
  • Melt DNA into single strands attached to a substrate.
  • RNA labeled with radioactivity or fluorescent dye.
  • The RNA and DNA will form a double-stranded hybrid molecule that is able to be detected.

Gel Electrophoresis of DNA

In an electrical field, samples containing different size DNA fragmentswill migrate through a gel containing pores of fixed sizes.

This is done using the charge properties of DNA. DNA in gel is visualized with a UV-visible dye.

Southern Blots

• Used to identify which clones contain a given DNA sequence.
• Cut DNA with restriction enzyme, separate fragments on a gel.

Polymerase Chain Reaction

• PCR requires two primers:
  • Primers are short, single-stranded sequences. One end complementary to 5’ end and the other complementary to the 3’ end of a DNA fragment.
  • DNA is mixed with DNA polymerase.
  • Primers are annealed and DNA is denatured
• Steps in PCR Cycle:
  • Denature DNA
  • Lower the temp to 45-65 C. Anneal primers to 5’ and 3’ ends of DNA.
  • Adjust temp to 65-75 C. Add DNA polymerase and dNTPs for strand extension.
  • Repeat using thermocycler
• One of the most widely used in genetics and molecular biology.
  • Used to screen for mutations involved in genetic disorders.
  • Allele-specific probes used for diagnosing genetic disorders.
  • Amplifies DNA from small samples for forensic analysis.

DNA Sequencing

• Dideoxynucleotide sequencing
  • Was most common method.
  • Dideoxynucleotide: Deoxynucleotide with a hydrogen at 3\('\) instead of an OH.
  • Insertion of a dideoxynucletide in a new strand of DNA causes the synthesis to terminate (No OH group to attach to).
  • Shortest chain (lowest on gel after electrophoresis) represents the first termination. The gel is read from bottom up.
  • Automated sequencing using Sanger method that uses nucleotides labeled with fluorescent dyes. Software aided.

High Throughput Sequencing

• Done with Sanger sequencing.
• In 2008, NHGRI started research program to drive cost down to $1000 per genome in 10 years.
• Several methods have been developed since then, and the cost is at the $1000 level.

Creating Transgenic Animals

• To study human disease, hundreds of transgenic animal strains carrying mutant alleles for genetic disorders have been created.
• These allow researches to study early how the disorders manifest, i.e. the molecular basis and test drug therapies.
• Mice with HD (Huntington’s disease) with 48 or 89 CAG repeats had paralleling events (brain cell counts dropped) with humans.