GENETIC TEACHER

DNA Template Strands consist of (Sense Strand= 5’ to 3’ direction) and (Antisense Strand= 3’ to 5’ direction), After transcription process the antisense strand convert to 5’ to 3' direction for RNA template as the same sequences and polarity as sense strand of DNA template #geneticteacher

A codon can be interrupted by an intron in one of three places Phase 0, Phase 1 and Phase 2, and all of these phases called Intron phase, which are important for splicing in transcription level, and in translation mechanism start codon is ATG, while stops codons are TAA, TAG and TGA #geneticteacher

Eukaryotic gene structure consist of Upstream intergenic region and downstream intergenic region and transcribed region involved 5-UTR (5-untranslated region) and 3-UTR (3-Untranslated region) and exons and introns, therefore 3% of eukaryotic genome is coding, while, over 85% of prokaryotic genome coding, Splice sites have donor (almost always Guanine and Thymine) and acceptor (almost always Adenine and Guanine), Interphase between exons and introns called donor site, while interphase between introns and exons called acceptor site, Cap region nucleotides (represent first nucleotide of gene structure), TATA box nucleotides (located in approximate 25 base pair in upstream region), CCAAT box nucleotides (located in approximate 100 base pair., in upstream region), GC box nucleotides (located in approximate 200 base pair in upstream region) #geneticteacher

Organisms respond to their environment via information from sensory input and changes in gene expressions in dynamic - immediate and transient modification of DNA program, the natural selection depend upon replication- mutations in DNA-RNA and Protein levels #geneticteacher

Gene is a unit of inheritance it carriers the information of polypeptide and for structural of RNA molecule, Similar genes have similar regulators under certain conditions, gene known locus or a position on a chromosome, allele mean alternate form of DNA at specific locus or gene on the chromosome, Each individual inherits two copies of DNA from maternal and parental, Homozygous allele is identical allele, Heterozygous is different allele, Different gene called allele #geneticteacher

DNA digest at 37 degree Celsius like body temperature because body temperature is optimal for most other enzymes, If the temperature too hot enzymes may be denatured and killed, if the temperature too cool enzyme activity become lower and required longer time for digestion, Each enzyme digest or cut DNA at specific sequence called restriction site and each recognition site have 4 or 6 base pair called palindromic sequences #geneticteacher

Cell cycle phases: 1- G1-Phase, primary growth phase and longest phase, 2- Synthesis-Phase, replicate DNA, 3- G2-Phase, organelles replicate and microtubes organized, 4- Mitotic-Phase, subdivided into (Prophase, Metaphase, Anaphase and Telophase), 5- Cytokinesis-Phase, divided cell separated to two new cells #geneticteacher

Mitosis Divisions results 2n diploid cells, while meiosis divisions results 4n haploid cells, so mitosis divisions originate genetically identical cell while meiosis division originate genetically different cells, hence, mitosis divisions occurs in somatic cells and meiosis divisions occurs in sexual cells #geneticteacher

Mitotic Catastrophe, is a malfunction of any checkpoints at G1-Phae, S-Phase, G2-Phase and M-Phases of the cell cycle, so it defines as the failure to arrest the cell cycle before or at mitosis resulting abnormal chromosome separation, which leads to generate aneuploidy cells (cell containing abnormal chromosome numbers), thus, mitotic catastrophe may be regarded as one of the mechanisms contributing tumor cell development #geneticteacher

Cell cycle interrupted in three checkpoints: G1-Chekpoint, makers the decision about whether the cell should divide and enter S-phase or no, but some cells never pass this point and are said to be in G0-Phase or Non-dividing, G2-Checkpoint, leads to mitosis, 3- M-Checkpoint, occurs during metaphase and trigger the exit process of the M-Phase and entry to the G1-Phase, so for many cell the G1Chekpoint is the most important, and if the cell receives go a head signal, it will usually complete the S-Phase G2-Phase and M-Phase , while, if the cell doesn’t receive the go a head signal, it will exit the cycle, switching into non-dividing state called G0-Phase, so checkpoints allow cell to respond internal and external signals #geneticteacher

The restriction checkpoint or point of no return, is the most important checkpoint in cell cycle, at this checkpoint, the cell self evaluated its won replicative potential before deciding to either enter the synthesis phase and the next around of cell divisions or to retire and leave the cell cycle #geneticteacher

Through the cell cycle several internal quality control mechanisms or checkpoints represented by biochemical control transitions between cell cycle stage, and the cell cycle stops at several checkpoints, checkpoints monitor cells through the cell cycle in response to intracellular of environmental signals #geneticteacher

Cell cycle is crucial process that increase the number of cells permits renewal of the cell populations and allows wound repair #geneticteacher

The cell cycle represents all events that take place in the period of time from the beginning of one cell divisions to the beginning to the next, and mature cells take longer than those embryonic tissue to grow #geneticteacher

The cell cycle incorporate two principle phases (Interphase and Mitosis Phases), Interphase represents continuous growth of the cell and subdivided into three phases, G1-Phase, usually longest and most variable phase and it begins at the end of the mitosis phases, Moreover, during G1phase the cell gathers nutrients and synthesis RNA and proteins necessary for DNA synthesis and chromosome replication, while, in S-phase the DNA of the cell doubles and new chromatids are formed, whereas, in G2-phase the cell examines its replicates DNA in preparation of cell divisions, so this phase called period of cell growth and re-organized of cytoplasm organelles before entering mitotic cycle, However, Mitosis phase takes place in several stage by separating two identical daughter cells #geneticteacher

Replicated chromosome consist of two strands of identical chromosomal materials called chromatids, therefore, cell cycle is a theoretical concept that defines the state of the cell divisions #geneticteacher

Mitotic Phase has two stages: 1- Mitosis Phase, the divisions of the nucleus, 2- Cytokinesis Phase, the division of the cytoplasm #geneticteacher

Stages of Interphase in mitosis divisions: 1- G1-Phase (Gap-1) the cell grows, protein synthesis and metabolic activities, 2- S-Phase, the cell replicates its DNA, 3- G2-Phase (Gap-2), the cell produced organelles and materials for mitosis divisions #geneticteacher

In eukaryotic, the cell cycles has four stages for cellular growth like protein synthesis, metabolic activities and DNA synthesis #geneticteacher

In prokaryotic, the cell grows, duplicates its DNA and divided by pinching in the cell membrane #geneticteacher

The goal of cell cycle is to produce two daughter cells, each containing chromosomes identical to those of the parent cell in about 24 hours #geneticteacher

Cell cycle represent self regulated sequence of controls cell growth and cell divisions #geneticteacher

Chromosome found in cell nucleus and sections of chromosome called genes and each chromosome has 1000 genes approximately, hence, chromosomal packaging DNA allowing efficient distribution of genetic material during cell divisions #geneticteacher

Applications of PCR are: 1- Study cloning and sequencing, 2- Monitor gene expression, 3- Diagnose genetic disease, 4- Study germplasm characterization for DNA fingerprinting, 5- Screen mutations, 6- Detect pathogenesis, 6- Measure genomic response in selection, 7- Make studies in bioinformatics #geneticteacher

Typical Programs of PCR Procedures: 1- Denaturation Phase (Denature template strands at 94 degree Celsius for (2 – 5) minutes, 2- Annealing Phase (Temperature variables for specific primers), so this phase is the most critical one for getting successful PCR reaction, 3- Extension Phase (generally at 72 degree Celsius) this is operating temperature for many thermos stable DNA polymerases #geneticteacher

Theoretical Yield of PCR 2 exponential cycles, so number of cycles depends on the number of copies template DNA and desired amount of PCR product (generally 20 to 30 cycles in sufficient) #geneticteacher

For 50 microliters reaction mixture in PCR tube are: 2 microliters Template DNA + 5 microliters 10X reaction buffer + 1 microliters for each primer (5 pico-mole) + 0.5 microliters Taq DNA polymerase + 39.5 sterile distilled water #geneticteacher

Procedures of PCR amplification in each cycle: 1- Heat double strand DNA for separating, 2- Hybridizations of primers be done, 3-Taq DNA polymerase and dNTPs and Magnesium's ions work for DNA synthesis from primers #geneticteacher

PCR primers types: 1- Universal primers (Amplifies all genomic DNA for instance), 2- Group specific primers (Amplify all denitrifies for instance), 3- Specific primers (Amplify just a given sequence) #geneticteacher

If you know the sequence targeted for PCR amplification, you know the size which the PCR primers should be annealing across #geneticteacher

The higher PCR primer melting temperature mean (PCR primer is more Specificity) #geneticteacher

PCR Primers: 1- Serves as a start site for DNA polymerase which extend in 5 to 3 direction, 2-should be (18 – 25) nucleotides in length that bind to one strand of the DNA and giving specificity, 3- Two primers are needed (One for each strand of DNA), 4- Calculating melting temperature of PCR primer depending on nearly identical for both primers, 5- Good primer design is critical to the success of PCR amplification, 6- Taq DNA polymerase cant not bind in the absence of PCR primers. #geneticteacher

PCR, is a technique sed to amplify DNA in minutes according to the following steps: 1- DNA is heated (denatured) to separated the strands of DNA at 95 degree Celsius, 2- Primers (short strands of DNA target specific regions of DNA for replication) by hybridize (Anneal) with the separated strands of DNA at 55 to 65 degree Celsius, 3- DNA polymerase directs the rebuilding of the double stranded DNA molecule using Taq DNA Polymerase and dNTPs at 72 degree Celsius (Repeated for 30 -40 times) to generate millions copies of double strand DNA, 4- Each strand of DNA used as a template to create replicate that permits doubling of the number of target molecules with each cycle of heating and cooling #geneticteacher

Component of PCR contains: 1- Target DNA (Contains the sequence to be amplified), 2- Primers (Oligonucleotides that define the sequence to be amplified), 3- dNTPs (DNA budling blocks, maintain pH and ionic strength of reaction solution suitable for activity of enzyme), 4- Thermostable DNA polymerase (Enzyme that catalyzes PCR reaction), 5- Magnesium ions (Cofactor of enzyme) #geneticteacher

PCR is in vitro reaction of DNA replication, identifying genomic DNA by means of amplifying by using specific primers compatible to DNA of interest #geneticteacher

Taq DNA polymerase, is an enzyme responsible for copying sequences starting at primer from single strand #geneticteacher

Thermostable DNA polymerase or Taq DNA polymerase, isolated from thermophilic bacteria (Thermus aquatics) , found in high heat temperature in hot springs in yellow stone national forest, so, it amplifies DNA from primers by polymerase chain reaction in presence of magnesium ions #geneticteacher

Bystander DNA not recognized by primers, while, target DNA contain primer recognition sequences #geneticteacher

In native gel conditions., use polyacrylamide gel electrophoresis as a support without denature, so proteins stays in original conformation for protection from oxidation, therefore movement conditions depends on intrinsic charge and hydrodynamic size, additionally., mechanism of polyacrylamide gel electrophoresis of native protein similar to SDS-PAGE #geneticteacher

Coomassie brilliant blue use to detect about 1 microgram of proteins in normal bands, while silver stain consider 100 times sensitive than Coomassie blue stain and use to detect 10 nano grams of proteins #geneticteacher

Running buffer in SDS-PAGE protocol have pH 8.3 (12 grams Tis Base + 57.6 grams Glycine + 4 grams SDS) dissolved in 4 liters distilled water, pour in migration tank and run the migration at 200 volts for 30 to 40 minutes #geneticteacher

Ethidum bromide and gel-red stains are preferred in DNA detection, while Coomassie brilliant blue and horse radish peroxidase are preferred in protein detection #geneticteacher

Methods of proteins detections: 1- Coomassie blue staining, 2- negative staining, 3- silver staining, 4- fluorescent staining, 5- fluorescent labelling, 6- radioactive labelling including (X-ray film., phosphor-imager plates., and stable isotope labelling) #geneticteacher

Disrupt SDS-PAGE buffer (10% SDS + Glycerol + Betamercaptoethanol + 1M Tris-HCL with pH 6.8 + Bromophenol blue) heat at least 95 degree Celsius for 4 minutes, (Tris-HCL, provide appropriate pH), (SDS use as detergent to dissolve proteins and give them negative charge), (Glycerol, use to make samples sink into wells), (Bromophenol blue, consider a dye to visualize samples) #geneticteacher

Sample preparation of SDS-PAGE (40 microliters proteins sample + 10 microliters disrupted buffer) boil mixture 3 minutes at 99oC #geneticteacher

Polyacrylamide gel has a tight matrix, so it consider ideal for proteins separation, due to smaller pore size than agarose, because of proteins much smaller than DNA, so the average of amino acids= 110 Daltons and the average of nucleotide pair= 649 Daltons, hence 1 Kbp of DNA= 650 Daltons, while 1 Kbp of DNA encodes 333 amino acids= 36 kilo Daltons #geneticteacher

To prepare 4% stacking gel in volume 4 millimeters, add 2.7 millimeters distilled water + 0.67 millimeters 30% acrylamide + 0.5 millimeters 1MTris with pH 6.8 + 0.04 millimeters 10% SDS + 0.04 millimeters 10% ammonium per sulfate + 0.004 millimeters TEMED, while, to prepare 10% running or separating gel in volume 10 ml, add 4 millimeters distilled water + 3.3 millimeters 30% acrylamide + 2.5 millimeters 1.5M Tris with pH 8.8 + 0.1 millimeters 10% SDS + 0.1 millimeters 10% ammonium per sulfate + 0.01 millimeters TEMED #geneticteacher

Stacking gel must be 5% polyacrylamide to concentrate all proteins in one band to start migrating in running gel at the same time, however, separating or running gel must be 8 to 15% polyacrylamide to separate proteins based on their molecular weight #geneticteacher