Recombinant DNA technology AbdelMoneim_2023 (1).pdf

09/09/2023 GENETIC RECOMBINATION It is an in-vitro modification of the genetic coding materials by inserting a new gene (gene cloning). Isolated DNA segments from different species like bacteria,viruses or...

09/09/2023 GENETIC RECOMBINATION It is an in-vitro modification of the genetic coding materials by inserting a new gene (gene cloning). Isolated DNA segments from different species like bacteria,viruses or animal are ligated together to form new combinations. 3 g New Combinations 3 2 1 3 2 1 Nucleic Acid 4 2  09/09/2023 Requirements for Recombination Donor DNA that contains C the required gene (Insert). Recipient DNA (Vector or plasmid). Restriction enzymes. DNA ligase enzyme. Host cell for cloning [competent cell]. 5 Steps of gene cloning The required gene( insert) is cut from the donor DNA by the restriction enzyme (RE). The recipient DNA (Vector) is cleaved by the RE. Insert is ligated to a cloning vector (plasmid) by ligase enzyme and the resultant is called (recombinant plasmid). Transformation: The recombinant plasmid is introduced into the competent cells. 6 3  09/09/2023 7 Cloning Vectors These are vehicles used to carry and introduce foreign DNA fragments into a host cell. Types of cloning vectors: 1. Plasmids 2. Bacteriophages 3. Cosmids 4. Yeast artificial chromosome [A human- engineered DNA molecule used to clone DNA sequences in yeast cells.] 5. Animal viruses [e.g. AstraZenca vaccine P Chimpanzee adenovirus with S gene of SARS-CoV-2] ⑧ & 8 8888 8 ena 4  09/09/2023 Cloning using Bacterial plasmid Ideal cloning vector must be : As small as possible Well characterized (gene location and nucleotide sequence) Capable of autonomous (self) replication within the host cell [Ori: Origin of replication]. Possess Multiple Cloning Site [MCS] at a localized area (a single cleavage site for different RE or at least for selected RE) (Multiple Cloning Site/ Polylinker) Carry a selectable marker (Antibiotic resistance gene) 9 : 10 5  09/09/2023 Flow chart of cloning Cut the DNA of the target Cut the plasmid DNA Ligate the target and plasmid DNA Introduce the ligated DNA [plasmid and insert] into bacterial cell. Confirm that the bacteria has the plasmid with the insert. 11 Cut the insert or plasmid DNA by Restriction Endonucleases (RE) Definition: Restriction endonucleases are enzymes that recognize and restrict (cleave) specific nucleotide sequences at both strands of DNA ⑳ molecule. Source: They are obtained from a wide variety of bacteria, and a few are obtained from fungi and algae. N.B.: The bacterial DNA is methylated in a specific manner to be protected itself from the cleavage by their restriction endonucleases. * EcoRI………E.coli RI 12 6  09/09/2023 Excising of the target genes and plasmids fee RE action results in either0sticky ends (e.g. 88 ⑧EcoR I) or blunt ends (e.g. Sma I ). - --13 ↑ m 13 Both sticky and blunt ended DNA molecules can be->8 joined using ligase that can be isolated from E.coli infected with T4 phage. You can not determine the direction of the blunt end ligated DNA [insert] without making sequencing. 5 14 7  09/09/2023 Easy cloning ↑ The pGEM®-T Vector is a linearized vector with a single 3 ́-terminal thymidine at both ends. The T-overhangs at the insertion site greatly improve the efficiency of ligation of PCR products generated by certain thermostable polymerase [e.g. Taq polymerase] that add 3' A overhang. See - I PGEM B 15 Tag polymerase 16 8  09/09/2023 Host Organisms for Transformation Microorganisms commonly used as hosts for cloning experiments include some bacteria & yeast. Strains used for this purpose should be: 1- Free of any restriction enzyme activity that might degrade foreign DNA. 2- The nucleic acid of the host be easily separable from that of the cloning vector by physical or chemical means. 3- High metabolic rate and high replication rate 17 do Transformation The recombinant DNA enters into the host cell (competent cell) and proliferates. Normal E. coli cells [competent cells] are difficult to take up plasmid DNA from the medium. If they are treated with CaCl2, the transformation efficiency can be significantly enhanced. Even so, only one cell in about 10,000 cells may take up a plasmid DNA molecule. 18 * ach 9  09/09/2023 19 Detection and characterization of the recombinant DNA Detection of the inserted DNA by Southern blot Hybridization 58D = - - - Detection of the transcript of the inserted DNA 80 (mRNA) by Northern blot hybridization. Detection of expressed gene product by Western blot hybridization G I Structural method of characterization e.g. restriction fragment length polymorphism analysis (RFLP) ⑳ 20 10  09/09/2023 well RFLP 21 2- Phage cloning vector [Phagemid] It has high transformation efficiency, about 1000 times more efficient than the plasmid vector. Two bacteriophages namely, Lambda (λ) and M13 are used for cloning. The virus genome is 49 kb in length which can carry up to 25 kb foreign DNA. 22 - 11  09/09/2023 Bacteriophages are viruses that infect bacteria When a phage enters Ecoli, it makes either Lysogenic infection Phage DNA is integrated into bacterial chromosome and replicate with the replication of bacteria (unfavorable condition) Lytic infection Production of complete virus particles (100 virus/cell) with lysis of bacterial cell (in favorable condition) Lytic replication of λ DNA results in long multimeric DNA called concatomeres that consists of multiple copies of viral genome linked by specific nucleotide sequence called COS sites (Cohesive end site or Sticky ends) 12 nucleotide long. Ss 23 ↳ The COS site helps the phage to backage its content into the phage head during phage replication. It also to circularize the phage DNA when it is inserted into the bacterial cell 24 ⑤8 12  09/09/2023 Dobb Two λ proteins called Nu1, and A bind to COS sites and directs the insertion of DNA lying between the cos sites into a phage head. Once the DNA is inserted into the phage head, the phage tail is attached to it forming complete virion. N.B. Bacterial cell genome is not packed into a phage head as it does not contain any copies of COS sequences. 25 ** 26 13  09/09/2023 Phagemid Phagemid (Phage+Plasmid) is a plasmid that also has certain bacteriophage-like properties Phagemid vectors are plasmid having: plasmid replication origin allowing isolation of double stranded (supercoiled) a replication origin usually from a single stranded phage such as f1 (filamentous phage origin) which allows the plasmid to enter a single strand replication mode in which only one of the single strand is packaged into the virus particle when helper phage is added. Phage coat protein gene Selective marker gene 27 3. Cosmid The cosmid vector is a combination of the plasmid vector and the COS site which allows the target DNA to be inserted into the phage head. It has the following advantages: High transformation efficiency. The cosmid vector can carry up to 45 kb whereas plasmid and l phage vectors are limited to 25 kb. 28 % 14  09/09/2023 Applications of Recombinant DNA Technology I- Therapeutic applications: 1- Production of recombinant vaccines, e.g. hepatitis B vaccine, SARS-CoV-2 vaccine[COVID-19 vaccine] 2- Production of biological products of medical importance, e.g.: - hormones, - interferons, - interleukins, - monoclonal antibodies... etc. 3- Gene therapy. II- Diagnostic applications: a- Gene mapping of microorganisms DNA. b- Preparation of nucleic acid (N.A.) probes. C- Developing of diagnostic kits by synthesis of purified proteins. 29 15