DNA Cloning

Anotações:

• Produce a large number of identical DNA molecules in a bacteria.

1. Examples

   Anotações:

   • Human insulin genes Human growth hormone Tissue plasminogen activator Factor VIII (prevent haemophilia) Hepatitis B virus vaccine (basically surface protein antigens)
2. Vector DNA cloning

   Anotações:

   • 1. Restriction enzyme celaves double stranded DNA. 2. Ligation of DNA with cleaved plasmid. 3. Recombinant Plasmid is slammed into the cell. By CaCl2 at 4 deg and heatshocked at 42deg for 2 minutes. Either this or electroporation.
   1. Plasmids

      Anotações:

      • Selection: 1. Antibiotic Resistance 2. Enzyme Inactivation 3. Size of Insert
      1. pUC19

         Anotações:

         • Antibiotic resistance determine if the bacteria survive The colour determine if the bacteria had the betagalactosidase to break down the lactose, if there was, it means the plasmid did not bind properly Plasmids multiply as the bacteria grow.
   2. Bacteriophage

      Anotações:

      • Based on lambda phage. only a certain size of insert from 15-21kb is able to be cloned.
   3. Properties

      Anotações:

      • 1. Origin of Replication  2. High Copy Number of vector in E.coli cell. 3. Vector is Small 4. Multi-cloning site for restriction enzymes 5. Antibiotic resistance genes
3. Artificial Chromosomes
   1. Bacterial Artificial Chromosomes

      Anotações:

      • Similar to pUC19, different origin of replication. Able to accommodate up to 100,000 inserts.
   2. Yeast Artificial Chromosome

      Anotações:

      • Clone very large segments of DNA. 50,000 to 1000000 base pairs. They need to have a bacterial origin of replication and antibiotic resistance gene.
4. Alkaline Lysis

   Anotações:

   • 1. Cells are lysed by SDS 2. DNA made single stranded by NaOH. 3. DNA neutralized and only plasmid remains. 4. Precipitates removed by centrifugation 5. RNA lysed by NaOH and RNase.
5. Libraries

   Anotações:

   • Presence of the DNA sequence is logged in the libraries.
   1. Genomic DNA clone

      Anotações:

      • Contains every Human DNA sequences
   2. mRNA clones

      Anotações:

      • Only expressed sequence is derived from mRNA. mRNA is copied into DNA using reverse transcriptase. cDNA is the result, and it does not contain introns. Reverse transcriptase goes from 5 to 3 in synthesis. RNA is removed by alkali and resyntehsis again,
6. Identification of Recombinant DNA
   1. DNA hybridization

      Anotações:

      • Identification was  done by autoradiography. A labelled probe was inserted in the colonies. Those who can bind to the colonies such as 32P(radioactive)GATC can bind to the selected product.
      1. Process

         Anotações:

         • 1. Colony hybridisation: Done via Southern Blotting Process. 2. Plate of bacterial colonies are transferred to a membrane forming a replica of bacterial colonies 3. Alkali lysed and DNA is made single stranded 4. Probe inserted. 5. Washing away unhybridized probe. Autoradiography is used to determine desired sequence.
         1. DNA Probe

            Anotações:

            • Oligonucleotides are synthesized from machine and uses 5' end to do radioactive labelling
   2. Expression of protein product
      1. Process

         Anotações:

         • Almost the same as the DNA vector
7. Blotting
   1. Northern Blotting

      Anotações:

      • Determines and analyses RNA Process is same as southern blotting. Isolate RNA from cells Electrophorisis on Agarose Gel Blot into Nylon filter Hybridize with 32P DNA probe, determine size of DNA
8. Techniques

   Anotações:

   • DNA sequencing and PCR is one of the  the 2 techniques that depends DNA Polymerase. REcall, DNA adds on from 5' to 3'. Termination occurs when ddNTP is incorporated. Therefore it will not be extended.