Week 2- Nucleic Acids and Proteins PDF

NUCLEIC ACID AND 2 PROTEINS HISTORY OF MOLECULAR DIAGNOSTICS YEAR KEY EVENT 1865 MENDEL'S LAW OF HEREDITY 1866 JOHANN MIESCHER, PURIFICATION OF DNA 1949 SICKLE CELL ANEMIA MUTATION WAS FIRST STUDIED 1953 WATSON AND CRICK’S DNA STRUCTURE 1970 RECOMBINANT DNA TECHNOLOGY 1977 DNA SEQUENCING 1985 IN-VIT...

NUCLEIC ACID AND 2 PROTEINS HISTORY OF MOLECULAR DIAGNOSTICS YEAR KEY EVENT 1865 MENDEL'S LAW OF HEREDITY 1866 JOHANN MIESCHER, PURIFICATION OF DNA 1949 SICKLE CELL ANEMIA MUTATION WAS FIRST STUDIED 1953 WATSON AND CRICK’S DNA STRUCTURE 1970 RECOMBINANT DNA TECHNOLOGY 1977 DNA SEQUENCING 1985 IN-VITRO APMPLIFICATION OF DNA (PCR) 2001 THE HUMAN GENOME PROJECT 1865 – Gregor Mendel discovered that the traits of parents could be passed through to the children. 1953 – It is one of the most important biological discovery of the 20th century when James Watson and Francis Crick described the structure of DNA. 1985 – Kary Mullis discovered the in vitro amplification of DNA. He won a noble prize because of the discovery of the Polymerase Chain Reaction (PCR). NUCLEIC ACID One of the Macromolecules in our body together with the Carbohydrates, lipids, and Proteins The building blocks of DNA and RNA are made up of nucleic acids. Macromolecules constructed out of long chains (strands) of monomers called nucleotides. Each nucleotides have three functional groups: Nitrogenous base Pentose Sugar Phosphate groups Storage and transmission of genetic information. The main function of the Nucleic acid is to store and transmit the genetic information from the DNA to become Protein. Genotypic and Phenotypic characteristics of the organism. DNA is very important for the storage of genetic information of the living organism. TWO TYPES: Deoxyribonucleic acid (DNA) Ribonucleic acid (RNA) Nitrogenous bases have two types: Purine Have two rings fused together, one six-membered and one fivemembered, containing four nitrogen atoms. Guanine Adenine Pyrimidine Have only one six-membered ring with two nitrogen atoms. Cytosine Thymine - specific for DNA Uracil for RNA pentose sugar Sugar building block of the Nucleic acid These sugars have five carbon and are positioned from the first carbon up to the fifth carbon. Each carbon has their own function. Forms a glycosidic bond with the nitrogenous base, linking 1st carbon the sugar to the base. This bond is essential for creating the nucleotide structure. determines whether it is Deoxyribose or ribose 2nd carbon -OH (Hydroxyl group) - ribose; Oxygenated h+ -H (Hydrogen group) - Deoxyribose; Deoxygenated 3rd carbon Attaches to the phosphate group through a phosphodiester bond. This forms the backbone of nucleic acid chains (DNA or RNA). 4TH CARBON Maintains the ring structure of the sugar molecule, ensuring its stability. 5TH CARBON Forms a bond with the next sugar molecule in the chain, creating the polynucleotide backbone (RNA or DNA strand). NUCLEOTIDE NUCLEOSIDE PHOSPHORYLATED SUGAR + BASE SUGAR + BASE (W/O PHOSPHATE GROUP) DEOXYRIBONUCLEIC ACID (DNA) Primary function is to store genetic information. Usually found in Nucleus, and some (small amount) are found in the Mitochondria. Assembled in units of nucleotides that are composed of a phosphorylated ribose sugar and a nitrogen base. Nitrogen bases are attached to the Deoxyribose sugar which forms a polymer with the deoxyribose sugars of the other nucleotide through a phosphodiester bond. DNA STRUCTURE Double helical structure First Described by James Watson and Francis Crick Their molecular model of DNA was founded on previous observation of the chemical nature of DNA including the Diffraction analysis performed by Rosalind Franklin. Two DNA chains form hydrogen bonds with each other in a specific weight, because these hydrogen bonds between the nucleotide are the key to the specificity of most nucleic acid base test in Molecular Laboratory. DNA DOUBLE HELIX The sequences of the two strands that form the double helix are complementary Base pairing which follows Chargaff’s rule 1. ThE RULE STATES that the total number of purine bases (A + G) is equal to the total number of pyrimidine bases (C + T). This rule holds true for both strands of the DNA molecule, regardless of the organism. 2. This rule reflects the complementary base pairing in DNA, where adenine pairs with thymine through two hydrogen bonds, and guanine pairs with cytosine through three hydrogen bonds. The complementary strands are Antiparallel orientation, meaning the 5’ end of one strand binds with the 3’ end of the other strand. Because of how the DNA is being replicated. The formation of hydrogen bonds between two complementary strands of DNA is called hybridization. Hydrogen bonds: g-c = 3 h-bonds a-t = 2 h-bonds DNA replication 1. dna polymerase It's responsible for synthesizing new DNA strands by assembling nucleotides, the building blocks of DNA, based on an existing template strand. FUNCTIONS: Reads the existing DNA strand (template) and adds complementary nucleotides to the growing new strand. Ensures accurate copying by checking each added nucleotide for a perfect match with the template base. Has proofreading activity to correct any mistakes made during the copying process. Works with other enzymes and proteins to ensure efficient and accurate DNA replication. TYPE FUNCTION 1 repairs errors in existing DNA 2 not directly involved in the main DNA replication 3 major enzyme for replicating the entire DNA molecule 2. helicase This will unzip the DNA strand. The site where the strand are being unwound by the helicase enzyme is called replication fork. 3. single stranded This will bind to the template/ parent strand to prevent the rebinding of the complementary bases. 4. topoisomerase Placed in front of the replication fork. This enzyme will prevent the super coiling of the DNA during the unzipping of the complementary strand. 5. primase The primer will activate the DNA Polymerase 3. The DNA Polymerase 3 will start the adding of nucleotide bases to the daughter strand. 6. leading strand top strand; this strand can build from one continuous strand towards the replication fork. 7. lagging strand bottom strand; cannot build one continuous strand because there is lagging. there are gaps present. it is building away from the replication fork. 8. rnase h (polymerase 1 enzyme) During DNA replication, short RNA primers are used to initiate DNA synthesis. RNase H removes these primers after DNA polymerase extends the DNA strand. 9. DNA Ligase help to connect the two fragments forming a one continuous strands. 10. okazaki fragment is a short sequence that was formed in the lagging strand that will be connected together by the DNA ligase. RESTRICTION ENZYMES aka. molecular scissors A type of Enzyme used for the degragation of the DNA. exonucleases degrades the DNA from its end. Either in the 5’ or 3' very useful in proofreading a newly synthesized dna or removing a noncomplementary base by breaking the phosphodiester bond and replacing by a correct one. 2. endonucleases degrades the dna in the middle. useful in inserting a new sequence in the middle part used also to identify a recognition site of endonuclease TYPES: TYPE 1: RANDOM CUT TYPE 2: MOST IMPORTANT; MAKES A SPECIFIC CUT TYPE 3: NON-SPECIFIC CUT To ensure that the restriction enzyme will not cut it self. nucleic acid. Methyltransferase enzyme will add methyl group to the self nucleic acid. they are resistant to degragation so that when the restriction endonuclease attacks, it will only cut the invading nucleic acid and not your own nucleic acid. Most of these enzymes are isolated from bacteria. For each recognition site, it will make a specific cut depending on what enzyme you will use. Deoxyriboendonucleases or Endonucleases Break the sugar-phosphate backbone of DNA. Restriction enzymes Endonucleases that recognize specific base sequences and break or restrict the DNA polymer at the sugar-phosphate backbone dna ligase Catalyzes the formation of a phosphodiester bond between adjacent 3'-hydroxyl and 5'-phosphoryl nucleotide ends Nucleases degrade DNA from free 3'-hydroxyl or 5'-phosphate ends other dna metabolizing enzymes: Nucleases degrade DNA from free 3'-hydroxyl or 5'-phosphate ends methyltransferases catalyzes the addition of methyl groups to nitrogen bases helicases separation of the sugar-phosphate backbones in both strands recombination (sexual reproduction) Mixture and assembly of new genetic combinations Common for Humans Example is the Mendel's law, each generation of sexually reproducing organism is a new combination of parental genomes. Recombinant chromosomes are found in the children. Half of the traits are from the mother, and half are from the father. (Diploid) recombination (asexual reproduction) Genetic information in asexually reproducing organisms can be recombined in three ways: A. conjugation The Transfer of genetic material from one organism to one organism through direct contact. b. transduction passes the genetic material from the donor to the recipient through the use of carrier. C. transformation target sequence from the donor cell could be cut using the restriction enzyme, and that specific sequence will be inserted inside the chromosome of the recipient cell. plasmids Most plasmids are double-stranded circles of 2,000 to 100,000 bp (2 to 100 kilobase pairs) in size. Plasmids can carry genetic information Plasmids were found to be a source of resistant phenotypes in multidrug- resistant bacteria. They carry the Antibioticresistant gene. Very short sequence If an organism is an antibiotic resistance, it can passed the information from the plasmid from one bacteria to another through direct contact or Conjugation. RIBONUCLEIC ACID (rNA) Polymer of nucleotides similar to DNA Synthesized as a single strand rather than as a double helix RNA strands do not have complementary partner strand Nitrogen bases: Adenine Cytosine Guanine Uracil Single Stranded; smaller than the DNA Its function is to translate the DNA to become proteins (phenotypic characteristics) Most DNA are found in the Cytoplasm; mRNA is found in the Nucleus types of rna 1. Ribosomal rna The largest component of cellular RNA 80% to 90% of the total cellular RNA Important structural and functional part of the ribosomes, cellular organelles where proteins are synthesized Various types of ribosomal RNAs are named for their sedimentation coefficient (S) Three rRNA species in prokaryotes 16S 23S 5S rRNA species in prokaryotes Single 45S precursor RNA (pre- ribosomal RNA) Most abundant; and it has catalytic roles and structural roles 2. messenger rna In prokaryotes, mRNA is synthesized and simultaneously translated into protein. Prokaryotic mRNA is sometimes polycistronic Eukaryotic mRNA is monocistronic In eukaryotes, copying of RNA from DNA and protein synthesis from the RNA are separated by the nuclear membrane barrier Most important; it will transport information from DNA to Ribosome. mRNA Transcription: Constitutive Transcription Inducible or Regulatory Transcription 3. transfer rna Translation of information from nucleic acid to protein requires reading of the mRNA by ribosomes, using adaptor molecules or transfer RNA (tRNA) Relatively short, single- stranded polynucleotides of 73 to 93 bases in length MW 24,000 to 31,000 Responsible in carrying individual amino acid to the Ribosome wherein they will be joined together by peptide bonds to make that protein. It contains three letter nucleotides: UAG This UAG carries an amino acid molecule The mRNA should be complementary to the tRNA containing the specific amino acid 4. small nuclear rna functions in splicing in eukaryotes RNAs sediment in : range of 6 to 8S Found in the Nucleus only Product of RNA splicing. Splicing is happening to remove the unnecessary sequences in the mRNA 5. srna Untranslated RNA molecules 6. ncrna Non-coding RNA TRANSCRIPTION This is the copying of information from the DNA to mRNA because DNA can only store information, In order for this information to be utilized it must be transcribed and translated into protein. GENE EXPRESSION: A process of transcription and translation copying of one strand of dna into rna by a process similar to that of dna replication catalyzed by rna polymerase Three types of rna polymerase: pol 1 - non-coding rna pol 2 - most important; the one responsible for polymerizing rna pol 3 - non-coding rna The RNA polymerase uses one strand of the Double helix that will serve as a template for the synthesis of the RNA. About 10 base pairs of DNA would be unwind with the use of RNA Helicase to allow the DNA polymerase to work. The Primer will attach specifically where is the start of transcription. Specific part of the DNA only. It's the same process with the DNA replication proteins Products of transcription and translation of the nucleic acids They manifest the phenotype directed by the nucleic acid information Polymers of amino acids Proteins are polypeptides that can reach sizes of more than a thousand amino acids in length Most abundant macromolecule in cells amino acids Each amino acid has characteristic biochemical properties determined by the nature of its amino acid side chain. Grouped according to their polarity Determine the shape and biochemical nature of the protein. The molecule of an amino acid is composed of four different groups: Amine group side chain carboxylic acid group All of them are connected with the Carbon on its middle. Zwitter ion it can switch from positive or negative charge. But at physiologic pH, most of the amino acids are negatively charge. protein structure 1. Primary Structure The linear sequence of amino acids joined together by peptide bonds Once they are formed inside the Ribosome, they will form a long chain of amino acid called as the primary structure. Minor changes in primary structure will definitely have an effect (Disorder or Abnormality) Hgb S (Sickle cell anemia) is a product of amino acid substitution. ON the 6th amino acid, Valine is seen instead of glutamine. It is a genetic problem 2. secondary Structure The regular folding of regions of the polypeptide chain There is a folding or coiling because of the R-side chain. The R-side chain of each amino acid will interact with each other and formed hydrogen bond. Folded - -pleated sheet Coiled - a helix 3. tertiary Structure The 3D arrangement of all the amino acids in the polypeptide chain Is also important for protein function. This folds are specific with each other. The folding of a protein is specific as well. If there's any problem with the folding or changes with the R-side chain, there will be an abnormality on the folding of the Protein. Denatured protein - loss of tertiary structure; non-functional. 4. Quaternary Structure This is formed by the interaction of different polypeptide chains Groups of tertiary structure bound together to form either dimer, trimer, or tetramer, depending on how many tertiary structure are present. Oligomers - group of tertiary structure, they give complexity in an organism. Monomer - each component of protein; tertiary structure. chromosomes DNA double helix that carries genes Seen during cell division Creates identical copy of itself Centromere: The X shaped is rarely seen in a cell, they are usually in a threadlike only – Chromatin threads. during cell division, they will formed the X shaped chromosome. But at normal times, the DNA are found as long chromatin thread. chromosome formation During cell division, first, the DNA will coiled up and wrap itself around to the proteins called Histones. These Histones with wrapped DNA are called Nucleosome. And as the Nucleosome coils and twists up even more, it will produce the Chromatin fibre. Then this Chromatin fiber will later to be condensed and will coil and fold up even more until it will formed the Chromosome structure. This chromatin fibers begin to formed really tight loops and they coil up even more until they eventually formed the Chromatin structure. The Chromosomes are made up of DNA that is coiled up during cell division. KARYOTYPE Individual's collection of chromosomes Used to check for abnormalities GENOTYPE Genetic DNA composition of organisms PHENOTYPE Physical appearance KARYOTYPING the collection of 23 Pairs of Chromosomes. 22 pairs for the autosome and 1 pair for the sex chromosome And check for any abnormalities. Trisomy 21 (Down syndrome) There’s an extra chromosome present in the 21st chromosome of the patient.