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Bacteriophage lambda The bacteriophage lambda is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It has the structure of a typical bacteriophage: Head, Tail, long tail fibers. Viral DNA is a linear double-stranded 48.5 kbp with 12 base pair "stic...

Bacteriophage lambda The bacteriophage lambda is a bacterial virus, or bacteriophage, that infects the bacterial species Escherichia coli. It has the structure of a typical bacteriophage: Head, Tail, long tail fibers. Viral DNA is a linear double-stranded 48.5 kbp with 12 base pair "sticky ends" at both ends. These ends are complementary sequences and can hybridize with each other (this is the cos sit: Cohesives sites). Bacteriophage lambda as a cloning vector The use of lambda phage as a vector is essentially done according to two strategies: i. Insertional vectors: the insert DNA is cloned into the MCS, which in this case resides within the lacZ coding sequence. The cloning capacity is 13 kpb. ii. Replacement vectors (ou substitution): there is a central stuffer for proper packaging of the vector, which will be “swapped” with the insert DNA. The cloning capacity is 23 kpb. NB: Substitution vectors are preferred but are more complex to implement.  Bacteriophage lambda as a cloning vector An insertional vector A replacement vector Bacteriophage lambda as a cloning vector  Advantages & limitations of using phage  as cloning vector Avantages -Relatively high cloning capacity (10 to 25 kbp) -Efficient infection, therefore rapid propagation Limitations does not support large insertions, more complex to implement (compared to plasmids). Cosmid as a cloning vector A cosmid is an artificial vector. It is a plasmid with one or two cos sites of the lambda phage. The presence of cos sites allows in vitro packaging of cosmid DNA into lambda phage particles. The insert size can be up to 45 kpb. Cloning into cosmids is similar to cloning in λ. It involves digesting exogenous DNA with a restriction enzyme, cutting the cosmid vector with a compatible restriction enzyme, combining the two, and ligating them. Advantages & limitations of using cosmids as cloning vectors Avantages -Cloning of large inserts (up to 45 kbp) -Infection process rather than transformation of E. coli therefore maintenance of a large number of recombinant phage particles. -The cosmid reproduces as a large plasmid and does not destroy infected bacteria. Limitations - Complex realization - DNA rearrangements can occur  Agrobacterium as a vector Hairy root disease Cane gall disease Crown gall disease Agrobacterium as a vector  Agrobacterium as a vector Ti plasmid of Agrobacterium Important: -Any DNA placed between the two borders can be transferred to plant cells - ~10% of plasmids are transferred to plant cells after infection  Binary vector system. The gene is cloned in-between the T- DNA borders of a broad host range plasmid. When mobilized to Agrobacterium this plasmid is able to replicate autonomously. The vir functions are supplied in trans by the plasmid. Co-integrate vector system. The cloned gene and a selectable marker (not shown) are cloned in pBR322. This plasmid is then mobilized to Agrobacterium. A single cross-over results in the co- integrate vector (black box indicating the pBR homology). The entire sequence between the T-DNA borders (wavy lines) which includes a pBR repeat will be transferred to the plant cell.  Binary Vector: Binary plasmid, its components and vir helper Ti plasmid SBMG: Selective plant marker gene, RES: Antibiotic resistance gene, OriT: Origin of transfer Bom region, RK2 Replication origin, which belongs to pRK2 plasmid Cointegrate Ti plasmid and its components (=integrative or hybrid Ti plasmids) MCS: Multiple cloning site, SBMG: Selective plant marker gene, RES: Antibiotic resistance gene, OriT: Origin of transfer, Col E1 Replication origin, which belongs to pCol E1 plasmid  Retroviral Cloning Vectors Retroviral vectors are proficient transfer systems for introducing foreign genes into target cells. Retroviral vectors are derived from RNA viruses, which are capable of reverse transcribing their genome into double-stranded viral DNA to enable stable insertion in the host genome. RNA viruses that are extensively used for virus vector engineering include murine leukemia viruses and lentiviruses. Retroviral Cloning Vectors  Retroviral Cloning Vectors Retroviral Cloning Vectors Principles of Retroviral Vector Gene Transfer  Shuttle Cloning Vector Shuttle vectors can survive in two different organisms, and include two origins of replication, one for each organism, and two genes for selection, one for each organism. Shuttle vectors can replicate in more than one organism. This allows the same gene to be expressed in different hosts. Shuttle Vector Artificial Chromosome Cloning Vectors Artificial chromosomes are DNA molecules of predictable structure, which are assembled in vitro from defined constituents that behave with the properties of natural chromosomes. ✓ Yeast artificial chromosomes (YACs), ✓ P1 artificial chromosomes (PACs), ✓ Bacterial artificial chromosomes (BACs) ✓ Human artificial chromosomes (HACs)  Yeast Artificial Chromosome (YAC) Yeast artificial chromosomes (YACs) are genetically engineered chromosomes derived from the DNA of the yeast. YAC cloning vector consists of two copies of a yeast telomeric sequence (TEL) (telomeres are the sequences at the ends of chromosomes), a yeast centromere (CEN), a yeast ARS (an autonomously replicating sequence where DNA replication begins), and appropriate selectable markers URA3. Multiple Cloning Site for cloning. The amount of DNA that can be cloned into a YAC is, on average, from 100 kpb to 2 Mpb. Yeast Artificial Chromosome (YAC) The YAC has two forms, a circular form for growing in bacteria, and a linear form for growing in yeast. The circular form can be manipulated and grown like any other plasmid in bacteria since it has a bacterial origin of replication and an antibiotic resistance gene. In order to use this in yeast, the circular form is isolated and linearized such that the yeast telomere sequences are on each end. This form can accommodate up to 2,000 kb of cloned DNA inserted into its multiple cloning site (MCS).  YAC vector cloning steps YAC vector limitations  Bacterial Artificial Chromosome (BAC) Bacterial artificial chromosomes (BACs) are designed for the cloning of large DNA insert (typically 100 to 300 kb) in E. coli host. BAC vectors contain a single copy F-plasmid origin of replication (ori). The F (fertility) plasmid is relatively large and vectors derived from it have a higher capacity than normal plasmid vectors. F-plasmid has F (fertility) factor which controls the replication and maintain low copy number. Also, conjugation can take place between F+ bacteria (male) and F- bacteria (female) to transfer F-plasmid via pilus. BAC Vector components Common gene components of a bacterial artificial chromosome are: 1) oriS, repE – F for plasmid replication and regulation of copy number. 2) parA and parB for maintaining low copy number and avoiding two F plasmids in a single cell during cell division. 3) A selectable marker for antibiotic resistance; some BACs also have lacZ at the cloning site for blue/white selection. 4) T7 and Sp6 phage promoters for transcription of inserted genes.  BAC vector cloning steps P1-derived Artificial Chromosome (PAC ) P1-derived artificial chromosome, or PAC, is a DNA construct derived from the DNA of P1 bacteriophages and Bacterial artificial chromosome. It can carry large amounts of about 100-300 kilobases of other sequences for a variety of bioengineering purposes in bacteria. In this system, vector DNA and DNA fragments to be cloned are ligated and packaged in vitro into phage particles that can infect Escherichia coli. The inserted DNA is more stable.  PAC Vector components Human Artificial Chromosomes (HAC) A human artificial chromosome (HAC) is a mini-chromosome that is constructed artificially in human cells. That is, instead of 46 chromosomes, the cell could have 47 with the 47th being very small, roughly 6-10 mega-bases in size, and able to carry new genes introduced by human researchers. Using its own self-replicating and segregating systems, a HAC can behave as a stable chromosome that is independent of the chromo- somes of host cells.  HAC Vector components The essential elements for chromosome maintenance and transmission are the following three regions: 1. The “replication origin,” from which the du plication of DNA begins, 2. The “centromere,” which functions in proper chromosome segregation during cell division, and 3. The “telomere,” which protects the ends of linear chromosomes. They are useful in expression studies as gene transfer vectors and are tools for elucidating human chromosome function. Grown in HT1080 cells, they are mitotically and cytogenetically stable for up to six months. Expression Vectors It is a plasmid or virus that is specially designed for expressing genes in a cell. It is a vector widely used for protein production. It has basic features of a vector like: (a) origin of replication (b) MCS (c) Regulatory elements: promoters, enhancers, termination sequence, initiation sites, stop codon, etc. (d) Selectable marker  Expression Vector Diagram Construction Methods for direct transfer of DNA into eukaryotic cells (non-viral methods)  Calcium Phosphate meditaed DNA transfer Calcium phosphate/DNA coprecipitation is a widely used method for the introduction of foreign DNA into cells. DNA and calcium phosphate are allowed to form a precipitate that is then added to cells in culture. The cells internalize the DNA, leading to the expression of the transfected genes in the cell. A mixture of the nucleotides, calcium, and phosphate buffer forms a precipitate that is taken up by the cells via endocytosis. Then, the nucleotides either escape the endosome or undergo lysosomal degradation, of which the latter might lead to reduced transfection efficiency. Successfully escaped nucleotides can then be expressed in the target cells. Protoplast Fusion Eukaryotic cells are fused with bacterial protoplasts harboring the recombinant DNA to be introduced.  Electroporation-mediated in vivo gene delivery Microinjection Microinjection is one of the physical methods of gene transfer used to introduce DNA or other genetic materials directly into a cell using a small glass needle or micropipette. This method allows efficient transfer and integration of desired genes into the host cell’s genome. It provides precise control over the delivery of desired materials making it a powerful tool in various research areas.  Lipososmes mediated DNA transfer =lipofection These hollow microscopic spheres of phospholipid can be filled with DNA or other molecules during assembly. The liposomes will merge with the membranes surrounding most animal cells, and the contents of the liposome end up inside the cell, a process known as lipofection. Although lipofection works reasonably well, it is rather nonspecific because liposomes tend to merge with the membranes of any cell. Gene gun-based gene transfer Gene gun-based gene transfer, also known as a method of particle bombardment or ballistic DNA transfer, is achieved by propelling DNA coated fine particles (~ 1 to 3 μm) directly against cells or surgically exposed tissues using a device with pressured helium as the driving force.  1. Nucleic Acid Extraction 1. DNA Extraction DNA extraction is a procedure used to isolate DNA from the nucleus of cells. Purpose of DNA Extraction To obtain DNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc…  1. DNA Extraction Isolating the genetic material (DNA) from cells (bacterial, viral, fungi, human, animal and plant) involves three basic steps: Rupturing of cell membrane to release the cellular components and DNA Separation of the nucleic acids from other cellular components Purification of nucleic acids 1. DNA Extraction The choice of a method depends on many factors: A. The quantity and molecular weight of the DNA. B. The purity required for application. C. The time and expense. Two main methods: ✓ Method 1:Phenol-Chloroform Extraction method ✓ Method 2: Solid-Phase Extraction 1. DNA Extraction Method 2: Solid-Phase Extraction  1. DNA Extraction Method 1:Phenol-Chloroform Extraction method ▪ This method is suitable for extracting DNA from various samples, including blood, suspension culture, and tissue. ▪ It produces relatively high yields and higher purity DNA than conventional extraction methods Steps: 1-Lysis of the Cell and non-nucleic acid cellular components Cells are treated with a lysis buffer typically containing denaturing detergents and chelating agents. EDTA (Ethylene diamine tetra-acetic acid) as a chelating agents, Inactivating DNAse enzymes and binds metal ions. SDS (Sodium dodecyl sulphate) as a detergent, helps in removal of lipid molecules and denaturation of membrane proteins. 2-DNA separation from proteins and cellular debris Separation Methods: Separate DNA From Crude Lysate Organic extraction Salting out By using ready kit (for whole extraction) b1. Organic extraction Traditionally, phenol: chloroform is used to extract DNA. When phenol is mixed with the cell lysate, two phases form. DNA partitions to the (upper) aqueous phase, denatured proteins partition to the (lower) organic phase. Phenol: Denatures proteins and solubilizes denatured proteins b2. Separation by Salting Out At high salt concentration, proteins are dehydrated, lose solubility and precipitate. Usually sodium chloride, potassium acetate or ammonium acetate are used. Precipitated proteins are removed by centrifugation. DNA remains in the supernatant. c-DNA precipitation - Precipitation of DNA: Absolute Ethanol is layered on the top of concentrated solution of DNA. - Fibers of DNA can be withdrawn with a glass rod. - Washing of DNA. - Desalt DNA: Most salts are soluble in 70% ethanol. Detailed Lab Protocol ▪ Besides organic methods, solid-phase extraction using a solid substrate, such as silica resins or beads, is another popular way to isolate DNA. ▪ Instead of using solvents to force DNA precipitation, this technique uses a simple lyse-bind-wash-elute process. Membrane based method By DNA clean-up kit More rapid and effective Use solid matrix to bind the DNA Wash away contaminants Elute DNA from column A spin column using a silica-based extraction method is used. This does not require the use of hazardous chemicals. Nucleic acids are attracted to the silica bead under high chaotropic salt concentrations. Steps: Limitations No technology is completely advantageous, and so the spin column is! Some crucial shortcomings, the spin column technique has. Here are a few. Loss of material: So as we increase the number of washing steps, we get less yield. Impurities Low yield: spin column kit can yield a maximum of 5 to 6 µG DNA while techniques like Phenol-Chloroform method can produce up to 10-15 µG of DNA or even more for blood. Fewer optimization options: for some hard samples like the plant, we need to perform optimization at every level. Costly: A single kit can perform only 25 to 50 samples. 2. RNA Extraction Isolation of intact RNA is essential for many techniques used in gene expression analysis such as: – Microarray analysis – Northern analysis – cDNA library construction – RT-PCR A number of RNA preparation technologies are widely available that can be classified into four general techniques: Organic extraction method Spin basket formats Magnetic particle method Direct lysis method RNA is not as stable as DNA and is susceptible to degradation by heat, RNases, and other enzymes. The effective RNA extraction protocol relies on these 5 things, ✓ Inactivation of RNase ✓ Stabilizing RNA ✓ Isolating RNA from DNA and protein. ✓ Precipitating only RNA ✓ Removing the DNA RNAs bind efficiently to the Basic MagPrep® Silica Particles at acidic conditions and in the presence of a chaotropic salt. Elution is obtained at a slightly alkaline pH (pH >8.0). MagPrep® Silica Optimized Acid-pH RNA extraction and Direct Lysis  2. Gel Electrophoresis Is a method of gel (made of agarose) electrophoresis used to separate and analyse DNA or RNA molecules by size. Agarose: is a liner polymer composed of alternative residues of D- galactose and 3,6-anhydro-L-galactopyranose joined by α (1→3) and β (1→4) glycosidic linkages. (A) Chemical structure of agarose. (B) The 3D structure of the AG, with pores of different size, observed by scanning electron microscope.  Electrophoresis: is the movement of charged particles under the influence of electric field. The electrophoretic migration rate of nucleic acids depends on: Ø Size of DNA molecules. Ø Concentration of agarose gel. Ø Voltage applied. Ø Conformation of DNA. Ø Buffer used for electrophoresis. ✓ The pore size in the gel is controlled by the initial concentration of agarose. ✓ The largest molecules will have the most difficulty passing through the gel pores. Because the phosphate group of each DNA nucleotide carries a negative charge, the DNA fragments migrate toward the positive end of the gel. ✓ Analyze the integrity of DNA samples. ✓ Calculate the size of DNA by the use of appropriate size markers. ✓ To see if your DNA fragments is pure and there is no contamination (?). ✓ Purification of nucleic acids fragments mixture How do you calculate molecular weight of DNA? 3. Molecular Hybridization  Molecular hybridization refers to the association that can take place between two single-stranded nucleic acids of complementary sequences and which leads to the formation of a double strand or duplex. This association is carried out by the establishment of specific hydrogen bonds (A=T, G=C) - a. Probe concept A nucleic acid probe is a nucleic acid molecule, antiparallel and complementary to a specific nucleic acid sequence, capable of recognizing by hybridization and labeling this sequence to allow its identification or isolation. A probe corresponds to a DNA fragment obtained directly from genomic DNA (gDNA) or from mRNA (cDNA) or synthetic oligonucleotides.  b. Preparation of the probes The marking of a probe is done in different ways depending on whether we are referring to radioactive or non-radioactive probes. Radioactive probes are labelled with the radioactive isotopes of sulphur, phosphorus or nitrogen for detection. Nonradioactive probes are the ones that are labelled with chemical tags or fluorescent molecules such as biotin, fluorescein and digoxigenin.