أسئلة الـ 11 ميكرو PPPM (قبل التعديل)

• These notes cover gene transfer and molecular diagnosis.

Gene Transfer

• It is the process of moving a piece of DNA (chromosomal or plasmid) from one cell to another.
• This happens using different means.

Types of Gene Transfer

• Vertical gene transfer involves the transfer of genetic material from a parental organism to its progeny.
• Horizontal (lateral) gene transfer happens when an organism transfers genetic material to another organism that is not its offspring.

Major Forms of Genetic Exchange in Bacteria

• Conjugation involves direct cell-to-cell contact.
• Transduction is mediated by a bacteriophage.
• Transformation involves the direct transfer of isolated DNA.

Conjugation

• It is a gene transfer form where two cells come in direct contact and DNA is transferred from a donor to a recipient.
• The donor cell contains an F plasmid.
• The donor plasmid contains a tra gene used to mobilize the plasmid.
• Conjugation primarily occurs in Gram-negative bacilli.
• A donor Gram-negative bacillus containing an F+ plasmid connects with an F- Gram-negative bacillus, with the F pili of the donor cell (F+) attaching to a receptor on a recipient cell (F-).
• The F pili contract the recipient cell for close contact, leading to canalization through the F pili between the cells.
• Once a canal forms, the F plasmid starts to mobilize one strand of its double-stranded DNA to the recipient cell.
• New double-stranded DNA is formed from the single strand in both the donor and recipient cells.
• The recipient now has the F plasmid and becomes an F+ cell, and can act as a donor cell.
• Conjugation also occurs in Gram-positive organisms, but is mediated by pheromones.

Transduction

• It is a form of gene transfer in which a bacteriophage (a virus infecting bacteria) mediates the DNA transfer.
• Types include generalized and specialized transduction.

Generalized Transduction

• It occurs with the lytic cycle of a bacteriophage, also known as a virulent phage.
• When the phage infects a bacterial cell and replicates, chromosomal DNA from the infected cell can be packaged into the phage head during assembly.
• Cell lysis and release of phages can infect other bacterial cells, transferring the chromosomal DNA to a new recipient cell.

Specialized Transduction

• Happens with temperate or lysogenic bacteriophages.
• Phage DNA integrates into the bacterial DNA at a specific region, called specialized transduction.
• When the integrated phage is excised along with a piece of bacterial chromosome and infects another bacterial cell, ittransfers this DNA to the recipient cell.

Transformation

• It is a gene transfer method involving direct uptake of DNA by a recipient cell either naturally or artificially in the laboratory.

Natural Transformation

• This is a rare occurrence (e.g., in Streptococcus pneumoniae).

Laboratory-Induced Competence

• Increasing effectiveness by adding calcium chloride solution and chilling on ice, then heat-shocking.
• In transformation by electroporation, a mixture of recipient cells and plasmids is exposed to an electrical field.
• This forms pores in the cell envelopes.
• Calcium chloride solution is added, chilled on ice, then heat-shocked.

Molecular Cloning

• It involves the process of reunion between two pieces of DNA, foreign pieces not typically found together, to produce a recombinant DNA molecule using recombinant DNA technology.

Steps in Molecular Cloning

• DNA extraction of the desired gene.
• Both the desired DNA and the vector are cleaved using the same restriction endonuclease enzyme (Type II REs) like EcoRI, resulting in "sticky ends" (terminal, single-stranded complementary sequences).
• Adding DNA ligase to ligate the desired gene into the vector.
• Formation of the recombinant molecule.
• Transformation of the recombinant molecule by electroporation or microinjection into a host cell (e. coli or yeast cell).
• Replication of the recombinant molecule and expression of the gene to produce the desired protein (e.g., insulin).

Molecular Cloning Principles

• To produce insulin using yeast cells or E. coli, extract DNA, then use a restriction endonuclease enzyme (like EcoRI) to cut the desired DNA and the vector at specific sites.

Importance of Molecular Cloning

• Used to produce vaccines.
• Used to produce drugs.
• Used to produce biologically important proteins.

Molecular Techniques in Diagnosing Microbial Diseases

• Polymerase chain reaction (PCR).
• DNA probe and hybridization.
• Blotting.

Polymerase Chain Reaction (PCR)

• It is an advanced technique used to generate many copies of a single DNA molecule.
• It is a common tool for diagnosing microbial and genetic diseases, for medico-legal purposes, for microorganisms typing, and for taxonomy and evolutionary studies.

DNA Probe & Hybridization

• A piece of single-stranded DNA or RNA, which is complementary to the sequence of interest and labeled for detection.
• The specificity of base pairing allows detection of unknown DNA or RNA.
• Binding is specific if complementary DNA is present, otherwise no binding occurs.
• Radioactive isotopes p32 are detected by Autoradiography.
• Fluorescence Rhodamine is detected by UV radiation.
• Biotin-Avidin and Digoxin-Antidigoxin are detected by enzyme product color.
• DNA Probe & Hybridization shares the same uses as PCR.

Blotting

• Probes are used for detecting DNA sequences by hybridization with a membrane, called blotting.

Types of Blotting

• Southern blotting: A method to detect single-stranded DNA that has been transferred to a nylon membrane.
• Northern blotting: A hybridization technique in which a DNA probe binds to an RNA target molecule.
• Western blotting: A detection technique in which a probe binds to protein target molecule.