Supervised by: Prof. DR. Zaki Abdul-Ghani Done by: Hussein Talal Ashour ID. 201117011
Recombinant DNA biotech. Allows the production of attenuated, vector and DNA vaccines
Gene therapy was one of the great potentials in pharmaceutical biotech. Which consists of the insertion of genetic material into cells to prevent, control or cure disease It encompasses repairing or replacing defective genes and making tumors more susceptible to other kinds of treatment
Enabling techniques
1) 2) Cutting and joining DNA molecules Cloning vectors
3)
4) 5) 6) 7)
Cloning vectors
A cloning vector is a small piece of DNA into which a foreign DNA fragment can be inserted.
The main type of vectors used for gene cloning are plasmid cosmids and bacteriophages, which are used according to the size of DNA fragments that need to be cloned into them.
Bacteriophages and cosmids are used for cloning of large fragments of DNA
Plasmids
Plasmids are small extrachromosomal DNA molecules that replicate independently using their own origin of replication.
Features that are generally found in plasmids and are used as cloning vectors:
1.
2.
Multiple cloning sites: Fragments of DNA that contain a number of different restriction sites, enabling the use of a choice of restriction enzymes for the cloning of DNA fragments.
3.
Plasmids
Plasmids
An ideal plasmid should 1. Be Small in size
2.
3.
4.
Contain large number of single restriction sites and high copy number (more than 10 copies per cell)
Bacteriophage
Most popular bacteriophage used is E.coli lambda bacteriophage which is made of tubular protein tail and a protein head packed with approx. 50kb of DNA.
After injection of the viral DNA into E.coli it can multiply and enter a lytic cycle leading to the lysis of the host cell and subsequent release of large no. of phage particles.
Alternatively, injection of the DNA can lead into a lysogenic cycle in which the phage DNA is integrated into the E.coli chromosome where its maintained until environmental conditions change and is then excised , entering a lytic cycle
Bacteriophage
Phage genome integrates by an attachment site (att) with a partially homologous site on the E. coli chromosome, where it replicates as a chromosomal DNA segment.
The interactions of two proteins, the cl genes expressed protein (by phage genome) and cro gene expressed protein (by E.coli chromosomes) decide between the events of the lytic and lysogenic pathways.
Bacteriophage
Following are the advantages of phage cloning system over the plasmids:
(i) DNA can be packed in vitro into phage particles and transduced into E. coli with high efficiency,
(ii) foreign DNA up to 25 Kb in length can be inserted into phage vector, and
rolling circle replication; 2. production of concatemers; 3. cleavage at cos site; 4. transcription and translation; 5. packaging. B - lysogenic cycle.
For transformation bacteria such as E.coli can uptake recombinant plasmid DNA by treating the cells with ice-cold CaCl2 until they reach a competent state in which they are ready to take up DNA.
In Electropoartion, DNA is introduced into both bacteria and eukaryotic cells. This technique is based on the induction of free DNA uptake by bacterium after subjecting it to a high electric field.
Genomic libraries are prepared by purifying total cell DNA and then making a partial restriction digest. Then such recombinant vectors transformed into suitable host cells and they are cultured in suitable selective medium for recombinant vectors. Selected clones are screened for specific genes and they are labeled and maintained as library.
The transcriptome is the set of all RNA molecules, including mRNA, rRNA, tRNA, and other non-coding RNA produced in one or a population of cells.
cDNA is produced from fully transcribed mRNA found in the nucleus.
cDNA is created from a mature mRNA from a eukaryotic cell with the use of an enzyme known as reverse transcriptase
Human genomic libraries can be constructed using restriction nucleases and ligase. A genomic library comprises a set of bacteria, each carrying a different small fragment of human DNA. For simplicity, cloning of just a few representative fragments (colored) is shown. In reality, all the gray DNA fragments will also be cloned.
Fragmented DNA coupled with vectors in the presence of DNA ligase. Recombinant vectors then transformed into E.Coli and cultured in selectable medium. From the colonies grown in the culture, DNA isolated from the vector and sequenced one by one. After sequencing, each colony with its genetic nature tagged and maintained as genome library.
Bacterial cells
Eukaryotic cell
1-genes are grouped into operons 1-genes are organize in single And hence transcribed together In transcriptional units interrupted A single molecule of mRNA by introns
2-whole process of transcription and translation take place in the cytoplasm 2-Transcription in nucleus, eukaryotic mRNA is firstly modified by the addition of a methylated guanine and then by the splicing of the introns. the mature mRNA is then exported into the cytoplasm where its translated into proteins
Plasmid libraries are used when: 1. Smaller insert size 2. Single gene is to be isolated
Vector
plasmid
Host
Specified by the origin Shotgun cloning of replication present cDNA cloning on the plasmid
Lambda bacteriophage
7-22
E.coli
Shotgun cloning
cosmids
25-45
E.cloi
Shotgun cloning
2- Immunological screening
Used when we need to isolate a gene coding for protein for which anti bodies available The success of this technique relies on the expression of the gene of interest
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2.
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DNA polymerase requires small fragments of double-stranded DNA to initiate DNA synthesis .
The PCR reaction uses special DNA polymerase that can withstand temperatures as high as 99 Celsius, working optimally at 72 Celsius and subsequently reducing the risk of mismatches that occasionally occur at lower temperatures.
Disadvatages
1. 2. 3. Require knowledge of DNA sequence Amplify only large fragments (up to 20 kb) Contamination may give false positive results
Thank you
References
1-Hugo and Russell's Pharmaceutical Microbiology 7th edition 2-http://en.wikipedia.org/wiki/Polymerase_chain_reaction 3-http://learnsomescience.com/wp-content/uploads/2011/05/PCRcycle.gif 4-http://biosiva.50webs.org/dna%20cl9.jpg 5-http://en.wikipedia.org/wiki/CDNA_library