2nd Semester Molecular Biology and Diagnostics (Nucleic Acids and Proteins) PDF

MODX311: MOLECULAR BIOLOGY AND DIAGNOSTICS TOPIC: NUCLEIC ACIDS AND PROTEINS 2nd SEMESTER | S.Y 2024-2025 TOPIC pairs is the unit regarding the SUBTOPIC...

MODX311: MOLECULAR BIOLOGY AND DIAGNOSTICS TOPIC: NUCLEIC ACIDS AND PROTEINS 2nd SEMESTER | S.Y 2024-2025 TOPIC pairs is the unit regarding the SUBTOPIC length of the DNA] SUB SUBTOPIC o Nitrogen bases come in pairs kaya base pairs Nucleic acids and proteins are macromolecules in our body. Proteins are the major macromolecule of the body. NUCLEIC ACID HISTORY OF MOLECULAR DIAGNOSIS One of the Macromolecules in our body together with the Carbohydrates, lipids, and Proteins YEAR KEY EVENT Macromolecules constructed out of long chains 1865 Mendel’s Law of Heredity (strands) of monomers called nucleotides. Johann Miescher; Purification of DNA Two Types: - They tried to isolate nucleic acid through alkaline lysis [Sodium - Deoxyribonucleic acid (DNA) hydroxide was used for them to - Ribonucleic acid (RNA) 1866 We should not just differentiate the two nucleic acid by extract DNA from the sample] - They know that there is genetic their strands, it is not reliable. material present in the cell [based - DNA is not always double stranded on the law of heredity] - RNA is not always single stranded 1949 Sickle Cell Anemia Mutation was first Differentiate them based on their structure studied - Nucleic acids are classified or defined as a polymer James Watson and Francis Crick’s DNA of nucleotide structure - Nucleotide is the monomer of nucleic acid 1953 - 3D structure [helical shape of the - Made up of 5-carbon sugar [pentose sugar] DNA] was introduced - Aside for the sugar, the nucleotide also contains 1970 Recombinant DNA technology nitrogen base DNA sequencing o The nitrogen base is attached to the first - Exact arrangement of the 1977 nucleotide or the nitrogen bases carbon of the sugar [adenine, thymine, guanine, o There are 4 possible nitrogen bases: cytosine, uracil] Adenine, Guanine, Cytosine, Thymine In vitro amplification of DNA (PCR) o In the RNA, instead of Thymine, there is - Discovered by Kary Mullis and he Uracil won a Noble prize in chemistry - Nucleotide are called dNTP [Deoxynucleotide 1985 because of his discovery of the triphosphate] Polymerase Chain Reaction [the o On the 5th carbon, there is the presence of technique/procedure; not the PCR phosphate group machine - On the 2nd carbon, there is another group, the The Human Genome Project hydroxyl group or the hydrogen group - It was started during the 1990s, but o This is where you will base on what type of it was just published during 2001 pentose sugar you have 2001 (not yet complete); In 2006, they released the complete version ▪ Deoxyribose – deoxygenated - They discovered that the DNA is [hydrogen group] 4.81 billion base pairs long [base 1|Page Transcribed by J.M.J.R.  ▪ Ribose – oxygenated [Hydroxyl group] st 1 carbon – nitrogen base 2nd carbon – hydrogen or hydroxyl group 3rd carbon – hydroxyl group [should be hydroxyl group always] We have 4 types of dNTP: [depending on what nitrogen base it was attached to] - dATP - dGTP - dTTP - dCTP if the sugar is unphosphorylated, you just have the sugar and nitrogen base, it will not be term as nucleotide. Instead, it is now called as nucleoside. - If there’s no phosphate, it is nucleoside To form the nucleic acid, it is a polymer of nucleotide - It is the strand of nucleotide [monomer of nucleotide attached to each other] o On the top of the nucleotide, there is phosphate group. On the bottom, there is hydroxyl group. The phosphate group and hydroxyl group of the other nucleotide will be bonded. It is called the phosphodiester bond. - The picture shows RNA. ▪ Phosphodiester bond is a covalent There are four (4) Nitrogen bases: bond seen in the phosphate group - Adenine and hydroxyl group of the previous - Guanine nucleotide - Cytosine If you read the DNA, the top part is phosphate and the - Thymine bottom part is hydroxyl group. There are two (2) types of nitrogen bases: - Phosphate is on the 5th carbon. Hence, it is the 5’ - Purine: made up of two or double ring structure end o Adenine and Guanine - Hydroxyl is on the 3rd carbon. Hence, it is the 3’ end - Pyrimidine: made up of a single-ring structure - If you read the DNA: 5’ to 3’ o Thymine and Cytosine 1 Purine/ 2 double ring and 1 single ring 2|Page Transcribed by J.M.J.R.  In pairing the nitrogen bases, we follow the Chargaff’s DEOXYRIBONUCLEIC ACID [DNA] Rule: one purine; one pyrimidine [one double-ring; one Macromolecule of carbon, nitrogen, oxygen, single-ring because the diameter of the DNA allows only phosphorous, and hydrogen atoms three rings (hindi pwededng sumobra)] Assembled in units of nucleotides that are composed of - The Adenine should bine with Thymine, and a phosphorylated ribose sugar and a nitrogen base. - Cytosine should bine with Guanine. - The nitrogenous bases are highly specific and they Nitrogen bases: held together in a certain pattern. - Adenine - Cytosine For the nitrogen bases to pair [usually, the DNA are - Guanine double-stranded, but not all the time], in Adenine and - Thymine Thymine, there are always 2 hydrogen bonds. In Cytosine and Guanine, there are always 3 hydrogen bonds. - Mas matibay si Cytosine and Guanine kasi mayroon silang 3 hydrogen bonds - In a DNA, there should be at least 40-60% Cytosine- Guanine bases Phosphate and Sugar are always on the outside; serves as the backbone of the DNA - Nasa loob ang nitrogen bases - Bond on the middle: hydrogen bonds - Bond in between nucleotides: phosphodiester bonds The DNA is in a helix form because the bases have hydrogen bonds, it is hydrophobic; and the sugar- phosphate backbone is hydrophilic - They are twisted or helical in shape because the backbone is hydrophilic and the bases are hydrophobic [to protect the bases] Their partner system is complementary [they are complementary base pairs] DNA STRUCTURE Double helical structure Described by James Watson and Francis Crick Diffraction analysis performed by Rosalind Franklin Helical structure of DNA results from specific sequence (order) of nucleotides in the strand, as well as the surrounding chemical microenvironment, Two DNA chains form hydrogen bonds with each other 3|Page Transcribed by J.M.J.R.  - All key players are enzymes except for the single- binding protein DNA POLYMERASE Enzyme responsible for polymerizing the nucleotide chains 5’ to 3’ [forward] – ang complementary ay 5’ to 3’ Template to determine which nucleotides to add to the [reverse] chain - Antiparallel orientation Reads the template in the 3ʹ to 5ʹ direction DNA DOUBLE HELIX The sequences of the two strands that form the double helix are complementary Follows Chargaff’s rule Antiparallel orientation - LUMA: 5’ ; BAGO: 3’ – enzymes like the newly made [3’] The formation of hydrogen bonds between two complementary strands of DNA is called hybridization. - Hybridization: when bonding occurs COMPREHENSION CHECK!!! DNA STRUCTURE - Anti-parallel, double stranded molecule - Sugar Phosphate backbone [hydrophilic] - Complementary base pairs joined by Hydrogen bond in the middle [hydrophobic] - Each strand has the potential to deliver and code for information Enzyme read: 3’ to 5’ - Length of DNA given in Base pairs - ALWAYS READ THE STRAND FROM 5’ TO 3’ HELICASE DNA REPLICATION This will unzip the DNA strand. The site where the strand are being unwound by the helicase enzyme is This is an enzymatic reaction called replication fork [where the separation occurs] The key players are the enzymes SINGE STRANDED BINDING PROTEINS - To start: we have the parent strand; if we want to This will bind to the template/parent strand to copy or replicate, the parent strand will be prevent the re-binding of the complementary bases. separated to replicate - Attach to the separated DNA so that it will not - From one double-stranded DNA, magiging two rehybridize with the original complementary double-stranded DNA during cell division [producing strand [no longer complementary] 2 daughter strand with the same genetic TOPOISOMERASE information] Placed in front of the replication fork. 4|Page Transcribed by J.M.J.R.  This enzyme will prevent the super coiling of the DNA - Okazaki fragment – is a short sequence that was during the unzipping of the complementary strand. formed in the lagging strand that will be connected PRIMASE together by the DNA ligase; incomplete DNA; pilit Very important to produce RNA primer. na bumabalik ‘yung primer sa 3’ end - An RNA [usually single stranded] primer - DNA Ligase will help to connect the two fragments [oligonucleotide: short] is a short strand of forming a one continuous strands. nucleotide that are specific/complementary to o put phosphodiester bonds in between the the 3’ end of the parent strand or template. Okazaki fragments to fill the gap of the [should be antiparallel as well; all should be fragments antiparallel when it comes to hybridization] - They will dictate where to start the replication. RESTRICTION ENZYMES - It will activate the DNA Polymerase 3 - RNA primer will be the 5’ end [dulo] which will DNA Degradation activate the polymerase 3 [maga-attach sa 3’ end 1. Exonucleases – it degrades the DNA from its end. of the primer] Either in the 5’ or 3’. Polymerase Enzyme: 2. Endonucleases – in the middle - Polymerase 1 or RNAseH o Also called as molecular scissors - Polymerase 2 ▪ Restriction enzymes - Polymerase 3 Attacks specific sequence of DNA POLYMERASE 3 o Three types of restriction endonucleases: Will start the adding of nucleotide bases to the ▪ Type 1 daughter strand ▪ Type 2 Once attached to the 3’ end of the primer, it will catalyze the addition of dNTP – will be added to the ▪ Type 3 growing chain of the DNA Nucleases: enzymes used to degrade nucleic acids - When the polymerase is adding, it should be [RNAses; DNAses] smart [complementary pairing only] - Nagddegrade either sa loob or labas or mismong - dGTP = dATP [not complementary] gitna ng DNA - dGTP = dCTP [complementary] and so on the copy is not the same with the template, it is complementary [kamukhang kamukha yung hiniwalayan na strand] DNA POLYMERASE 1 Also called as RNAseH Leading Strand: Top strand; the synthesis of the DNA is leading towards the replication fork Lagging Strand: Bottom strand; cannot build one continuous strand because there is lagging. There are gaps [pinaglagyan ng primer] present. It is building away from the replication fork - The primer with the help of DNA Polymerase 3 will synthesize new strand of DNA. - RNAse H (Polymerase 1 enzyme) will enter to remove the primer. - The primer will jump into a new site. Those gaps are Commonly used: 20 enzymes from the RNA primer and removed by the RNAse H. Palindromic: reads the same forward and backward There is a possibility for these fragments will bind. 5|Page Transcribed by J.M.J.R.  - Helicases- separation of the sugar-phosphate backbones in both strands RECOMBINATION (SEXUAL REPRODUCTION) Mixture and assembly of new genetic combinations Methyltransferases - Transfer methyl group to the DNA - Our DNA should be methylated because with the addition of the methyl group, the DNA will resist the restriction enzymes o Hindi mac-cut kung may methyl group o Example: A bacteriophage (virus that Genetic information in asexually reproducing organisms attacks a bacteria) will attack the bacteria can be recombined in three ways: releasing its nucleotide sequence inside. - Conjugation: direct transfer The bacteria will protect itself from the - Transduction: passes the genetic material through invading bacteriophage. The bacteria the use of carrier [indirect] containing restriction enzyme will cut the - Transformation: recombinant techniques; alter the nucleic acid of the Bacteriophage for it to nucleic acid or delete nucleic acids [deletion] stop its replication. o To ensure that the restriction enzyme will not cut it self-nucleic acid. Methyltransferase enzyme will add methyl group to the self-nucleic acid. Kailangan methylated na ‘yung nucleic acid, they are resistant to degradation so that when the restriction endonuclease attacks, it will only cut the invading nucleic acid and not your own nucleic acid. 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 PLASMIDS - Catalyzes the formation of a phosphodiester bond Most plasmids are double-stranded circles of 2,000 to between adjacent 3ʹ-hydroxyl and 5ʹ-phosphoryl 100,000 bp (2 to 100 kilobase pairs) in size. nucleotide ends Plasmids can carry genetic information Other DNA Metabolizing Enzymes: - Nucleases- degrade DNA from free 3ʹ-hydroxyl or 5ʹ- Plasmids were found to be a source of resistant phosphate ends. phenotypes in multidrug-resistant bacteria. - Methyltransferases- catalyze the addition of methyl groups to nitrogen bases 6|Page Transcribed by J.M.J.R.  Plasmids carry the antibiotic-resistant gene that is not seen in human Very short sequence and in circular form TYPES OF RNA RIBOSOMAL RNA (rRNA) RIBONUCLEIC ACID (RNA) The largest component of cellular RNA Polymer of nucleotides similar to DNA 80% to 90% of the total cellular RNA Synthesized as a single strand rather than as a double Important structural and functional part of the helix ribosomes, cellular organelles where proteins are RNA strands do not have complementary partner strand synthesized Nitrogen bases - Important for the protein synthesis - Adenine - Ribosomes is the site where proteins are produced - Cytosine Various types of ribosomal RNAs are named for their - Guanine sedimentation coefficient (S) - Uracil Three rRNA species in prokaryotes - 16S, 23S, 5S rRNA species in prokaryotes - Single 45S precursor RNA (pre- ribosomal RNA) The DNA is found in the nucleus and mitochondria - Mitochondrial DNA comes from the mother only. - DNA [genotype] needs to become phenotype - This is the central dogma: o DNA information is not allowed to exit the nucleus. o It will just make a transcript or copy in the form of messenger RNA [mismong photocopy of the information from the DNA] o mRNA will go to the ribosomes to become proteins o DNA → mRNA [transcription] o mRNA → protein [translation; inside the ribosomes] 7|Page Transcribed by J.M.J.R.  MESSENGER RNA (mRNA) The mRNA should be complementary to the tRNA containing the specific amino acid In prokaryotes, mRNA is synthesized and simultaneously translated into protein. SMALL NUCLEAR RNA (snRNA) Prokaryotic mRNA is sometimes polycistronic [multiple Functions in splicing in eukaryotes products] RNAs sediment in a range of 6 to 8s Eukaryotic mRNA is monocistronic [one product only – eukaryotes] OTHER RNAs In eukaryotes, copying of RNA from DNA and protein synthesis from the RNA are separated by the nuclear sRNAs membrane barrier ncRNAs Most important; it will transport information from DNA to Ribosome. mRNA has the same sequence that can be found from TRANSCRIPTION the DNA. Copying of one strand of DNA into RNA by a process mRNA will go to the cytoplasm specifically in the similar to that of DNA replication. Ribosome, because Ribosome is for protein synthesis. Catalyzed by RNA polymerase It is a single stranded molecule only. It is just a copy of Three Types of RNA polymerase one strand of the DNA. - pol I mRNA is always forward; you should copy on the - pol II reverse - pol III mRNA TRANSCRIPTION Constitutive Transcription [continuous] Inducible or Regulatory Transcription [when need arises; ex: during inflammation] TRANSFER RNA (tRNA) 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 31,00 8|Page Transcribed by J.M.J.R.  OTHER RNA-METABOLIZING ENZYMES Ribonucleases - Degrade RNA in a manner similar to the degradation of DNA by deoxyribonucleases - Classification: o Endoribonucleases o Exoribonucleases RNA Helicases - Catalyze the unwinding of double-stranded RNA 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 Proteome vs Genome AMINO ACIDS Each amino acid has characteristic biochemical properties determined by the nature of its amino acid side chain Grouped according to their polarity [polar and non- polar] Determine the shape and biochemical nature of the protein. Nucleotide: negative charge; Protein: both positive and negative charge - Zwitter ion: it can switch from positive or negative charge based sa pH o Blood pH: 7.35-7.45 [slightly alkaline] – the pH is dependent on the hydrogen bonds [mababa ang positive charges so, blood is negatively charged (physiologically speaking)] 9|Page Transcribed by J.M.J.R.  PRIMARY STRUCTURE The linear sequence of amino acids joined together by CHROMOSOMES peptide bonds DNA double helix that carries genes Seen during cell division Creates identical copy of itself - Centromere SECONDARY STRUCTURE The regular folding of regions of the polypeptide chain TERTIARY STRUCTURE The 3D arrangement of all the amino acids in the polypeptide chain DEFINITION OF TERMS KARYOTYPE: - Individual’s collection of chromosomes - Used to check for abnormalities GENOTYPE: - Genetic DNA composition of organisms PHENOTYPE - Physical appearance QUATERNARY STRUCTURE This is formed by the interaction of different polypeptide chains Example of Trisomy 21 10 | P a g e Transcribed by J.M.J.R.