DNA cloning and sequencing

Features of bacterial cloning

• Origin of replication

• Many restriction enzyme sites

  • Poly linker

  • Multiple cloning site

• Antibiotic resistance marker

• A way to select DNA insertion - blue-white screening

How is DNA inserted into a plasmid

• Restriction endonucleases = cut plasmids and DNA fragments

  • Recognition sites are usually palindromic

• DNA ligase

  • Joins sticky ends

Stages of DNA insertion into the plasmid

1. Digest the plasmid + insert with restriction enzyme

2. Compatible sticky ends join via H bonds

3. Recombinant plasmid allows propagation of DNA sequence

4. Selection process

   • Antibiotic markers selectively grow bacteria that have taken up the plasmid

     • Bacteria with the plasmid have the antibiotic resistance gene

     • Will survive and reproduce colonies when grown on agar plate containing the antibiotic

     • Produce clones

Source of genomic DNA

• Whole genome of DNA digested with restriction enzyme

• Create libraries of whole genome

Source of complementary DNA

• Generate after reverse transcription of mRNA

• Provides a copy of the coding sequence of a gene (no introns)

Cloning a PCR product

• Can use PCR to introduce a DNA fragment into a plasmid

  • Design restriction enzyme sites to the 5’ end of the primer

  • Digest PCR products with the enzyme before ligation into the plasmid

Outline Sanger sequencing

• Based on the use of dideoxynucleotides

  • Have no Oxygen on the 3’ OH

  • Chain elongation terminates

Mechanism of Sanger sequencing

1. Generate primer to bind

2. Polymerisation to extend DNA from primer

3. Have normal nucleotides in the mix

4. Have dideoxynucleotides (fluorescently labelled)

   • Identify which nucleotides have been added

   • Can separate bases in capillary electrophoresis - see which nucleotides have been addd where

Importance of Sanger sequencing

• Compare DNA sequences

• Evolutionary trees

• Highlight functionally importance of genes/proteins

• Identify disease-causing microorganisms

Uses of DNA sequencing

• Characterising specie’s genome

• Identifying specie

• Disease-causing mutations

• Verifying correct sequences of PCR/clonoing products