Recombinant DNA technology

1. Recombinant DNA technology

2. Isolation of genomic DNA from bacteria 1. Suspend bacterial cells in buffer. 2. Add Lysozyme [degrades peptidoglycan cell walls] Protein-denaturing agents (e. g. perchlorate) OR Protease [breaks down cellular proteins] Detergent (e. g. SDS) [solubilises membrane lipids] RNAse [degrades RNA]

3. 3. Add phenol:chloroform, mix gently and centrifuge. Aqueous phase DNA Denatured proteins lipids Organic phase

4. 4. Add ethanol to the aqueous phase. DNA precipitates in alcohol if the salt concentration is high. Total cellular DNA 5. Transfer DNA precipitate to new tube. Suspend in buffer solution.

5. Plasmids - stable extrachromosomal elements - usually circular ds DNA.

6. Plasmid isolation 1. Suspend plasmid-containing cells in buffer. 2. Treat with NaOH and SDS. This lyses cells and denatures DNA. 3. Neutralise lysate with potassium acetate [pH 4.3]. Plasmid DNA renatures. Long chromosomal DNA strands reanneal incorrectly to form an insoluble network. K+ ions precipitate protein-dodecyl sulphate complexes.

7. 4. Centrifuge. Chromosomal DNA network and protein sediment. The supernatant fraction contains plasmid DNA. 5. Transfer supernatant to a new tube. 6. Precipitate plasmid DNA with ethanol and redissolve finally in a buffer solution.

8. Molecular cloning -insertion of a DNA fragment into a vector and propagation of the recombinant molecule in a host organism. Vectors are usually derived from plasmids or phages.

9. Type II restriction enzymes give fragments suitable for cloning. e. g. Bam HI ---GGATCC--- ---CCATGG---

10. GATCC G G GATCC G G CCTAG G CCTAG CCTAG G G GATCC GATCC CCTAG G +

11. DNA ligases covalently join fragments of DNA. T4 ligase (from phage T4) is used in gene cloning. -joins fragments with cohesive ends or blunt ends. Substrates must have 5’ phosphate and 3’ hydroxyl groups.

12. G G p-GATCC GATCC CCTAG-p CCTAG G G G GATCC CCTAG G p-GATC CTAG-p Linearised vector DNA can be dephosphorylated. Alkaline phosphatase Dephosphorylated vector cannot ligate to itself but can ligate to insert fragments.

13. Recombinant DNA molecules can be introduced into bacterial cells by transformation.

14. Agarose gel electrophoresis is used to analyse fragments of DNA. Molecular weight markers Small fragments run faster.

15. Gene libraries A library is a set of clones representing the entire genome of an organism.

16. Genomic DNA is partially digested with Sau 3A. ---GATC--- ---CTAG---

17. Overlapping fragments are ligated to a Bam HI -cut vector. Recombinant molecules are propagated in a host strain.

18. Plasmid vectors have a low molecular weight, an antibiotic resistance gene (a selectable marker), unique restriction sites.

19. Bam HI Bam HI With pBR322 (4363 bp) Bam HI fragments can be cloned into the Bam HI site. Transformants with recombinant plasmids are AmpR and TetS. Bam HI Pst I Ampicillin R Tetracycline R ori

20. The number of clones required to represent the genome depends on (a) the size of the genome (b) the size of the insert in each clone. Large inserts are better, fewer clones are required. Plasmid vectors can only be used to clone fragments < 10 kb. Larger plasmids cannot be transformed into cells efficiently.

21. Phage l vectors have been developed. The central region of the l genome can be replaced with 20kb of foreign DNA.

22. N cI cro att int xis lysis Non-essential for lytic growth Head Tail DNA synthesis

23. 20 kb fragments of foreign DNA can be inserted between the L and R arm fragments. L R Bam HI Bam HI Recombinant phage genomes are packaged into phage particles in vitro. Phage efficiently inject large molecules into host cells.