Biology Notes (Term 3!)

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1 Biology Notes (Term 3!) RNA Definition Ribonucleic acid (RNA) is another nucleic acid that is chemically related to DNA Function Structure Serves an intermediate role in the expression of information contained within DNA mrna carries the genetic code for proteins rrna forms structural and functional components of ribosomes trna helps to incorporate amino acids into polypeptide chains Pentose sugar of RNA is ribose, rather than DNA s deoxyribose. Ribose has a hydroxyl group at the 2 position that 2 -deoxyribose lacks: RNA contains the pyrimidine uracil (U) in place of thymine GENETIC CODE FEATURES Feature Explanation Contains 3 ribonucleotide letters in a codon Unambiguous Degenerate Contains start and stop codons Commaless Universal Each group of three ribonucleotides, called a codon, specifies one amino acid; thus, the code is a triplet. A two-letter code would provide only 16 unique codons (4^2), which is insufficient. A three-letter code would provide 64 unique codons (4^3), and is much simpler than a four-letter code. Each codon specifies only a single amino acid A given amino acid can be specified by more than one codon, and this is the case for 18 of the 20 standard amino acids They are necessary for initiation and termination of translation. Start codon codes for the amino acid Met, and 3 codons are stop codons: UAA, UAG and UGA. Once translation of mrna begins, the codons are read one after the other, with no breaks Same code used for many living organisms Page 1 of 8

2 Transcription and Translation Central Dogma: DNA ----transcription----> RNA ---translation----> PROTEIN Transcription Steps Translation RNA polymerase recognizes and binds to the specific promoter sequence and initiate transcription RNA polymerase moves in the 3 to 5 direction along one strand of DNA (= template strand), unwinding it as it goes to synthesize RNA in the 5 to 3 direction. Complementary bases are assembled (U instead of T). Termination, where RNA polymerase encounters a transcription terminator in the DNA (to signal the end of transcription), releases the completed mrna, and dissociates from the DNA Initiation Elongation Termination The ribosome binds to the mrna Amino acids (carried by trnas) are incorporated into the polypeptide chain. The anticodon (trna) attaches to the mrna sequence at the A site, and then moves to the P site, where it is released The trna, polypeptide chain and ribosomes are released from the mrna when it encounters a stop codon Notes are so boring here are some pictures for you (Transcription on the left, Translation on the right) QUESTION TYPE: Name 2 differences between transcription and translation. Transcription Difference Translation Nucleus Location Cytoplasm DNA What sequence does it read! mrna mrna sequence Result Amino acid sequence/a protein It needs RNA polymerase! What is needed It needs a ribosome! Page 2 of 8

3 Genetic Mutations QUESTION TYPE: Usually will give you some sort of genetic disease e.g. cystic fibrosis, a normal and a mutant DNA, and asking you to transcribe and translate it. so the whole list of possible things they will ask you to do to DNA is shown below: Strand Name How it looks like (EXAMPLE) Note Non-template OR Coding Template OR Non-coding 5 TAGTAGAAAGCGCGC 3 3 ATCATCTTTCGCGCG 5 Reverse the 5 and 3, and write down the matching bases of the coding strand. mrna 5 UAGUAGAAAGCGCGC 3 Just replace all the T in the coding strand sequence to U :) trna 3 AUCAUCUUUCGCGCG 5 Just replace all the T in the template strand sequence to U :) Amino Acid OR Protein (i m lazy to read this sequence but you should know what this is!) Take extra precautions to look at the table that they have given you! APPROACHING THE QUESTION: (1) Open your eyes really big and look at the translation table they have given you. There are 3 types of tables, DNA codon table, mrna codon table and trna codon table. These 3 tables have the same function - translating a strand into the amino acid sequence but they translate from different strands! QUESTION TYPE (FOLLOW-UP): Afterwards, they will ask you to identify the present and its effect on the protein produced or something of that sort. APPROACHING THE QUESTION: (1) Find out which base(s) have been changed in the mutant strand, and translate the strand to the amino acid sequence (for it is only then you will know if the has any effect on the protein produced. Usually they will get you to translate it to the protein form anyway :) (2) Identify the type of present as explained below: Type What it essentially is Example Point Insertion or deletion Inversion Missense Also called Single-Base Substitution. A single base is substituted for another, and this can be either a transition : (purine -> purine OR pyramidine -> pyramidine) or a transversion : (purine -> pyramidine or vice versa) A base is either inserted or lost from the DNA sequence. Segment of nucleotide sequences separates from the allele and rejoins at the original but it is inverted. Caused by point -> the new nucleotide alters the codon so as to produce an altered amino acid in the protein product. From GCG to GGG (transversion) From GAG to GGG (transition) From GCG to GCTG (insertion) From GCG to GG (deletion) From GCGTATCG to GCTATGCG From CGC (Arg) to CAC (His) Page 3 of 8

4 Nonsense Silent Frameshift Caused by point -> the new nucleotide changes a codon of the DNA coding strand that specified an amino acid to one of the STOP codons (TAA, TAG, or TGA), thus translation will stop prematurely. The causes no change in the product and cannot be detected without sequencing the gene (or its mrna). Caused by insertion/deletion of bases, all subsequent codons downstream from the site are altered. From GCGAGA to GCGTGA (TGA is stop codon) From TCT (Ser) to TCA (still Ser) From GCG TAT CGC to GCT GTA TCG C WRITING YOUR ANSWER: (A) If it is a point : This is a point of the (state position e.g. 15th) nucleotide. This is also a (transversion/transition), as (guanine/cytosine/adenine/thymine) is replaced with (guanine/cytosine/adenine/thymine), thus altering the codon from (normal codon) to (changed codon). If missense: This results in a missense, where the amino acid (changed amino acid name) is coded for instead of the amino acid (original amino acid name), hence producing in a non-functional protein. If nonsense: This results in a nonsense, as (changed codon) is a premature stop codon, causing translation to be stopped prematurely, hence producing a truncated non-functional protein. If silent: This results in a silent, as the amino acid (name of amino acid coded by changed/original codon) is still coded for, hence, the protein produced is still functional. (B) If it is an insertion/deletion: This is an insertion/deletion of the (state position e.g. 15th) nucleotide. This alters all subsequent codons downstream from the site, hence producing a non-functional protein. (C) If it is an inversion: This is an inversion as the nucleotide to nucleotide is inverted. Explain missense/nonsense, producing a non-functional protein. TYPES OF MUTAGENS: Mutagens work by modifying nucleotide bases and breaking the phosphate backbone. Type Chemical Explanation/Example Base analogs: Chemicals that are similar to nucleotides and are mistakenly used in the formation of new but defective DNA Intercalating agents -Chemicals that interact and insert directly into the DNA of cells e.g. Ethidium bromide Other chemicals may alter the structure and pairing of bases in a DNA strand Radioactive Biological Gamma rays, Ultraviolet light Many viruses contain DNA that insert into the chromosomes/genome of human cells e.g. Human Papilloma Virus WHY IS MUTATION IMPORTANT: The ultimate source of all genetic variation is. Mutation is important as the first step of evolution because it creates a new DNA sequence for a particular gene, creating a new allele. Page 4 of 8

5 PLASMIDS & RESTRICTION MAPPING Structure of plasmids: Most plasmids are circular molecules, and consist of an origin of replication, a selectable marker and a multiple cloning site (MCS). Useful plasmid cloning vectors have the following properties: 1. Low molecular weight (makes plasmids more resistant to damage during plasmid extraction or manipulation) 2. Selectable marker (resistance to a particular antibiotic, typically) 3. Multiple cloning site (MCS - this DNA region contains unique recognition sequences for a variety of restriction enzymes) QUESTION TYPE: Restriction mapping of some plasmid. APPROACHING THE QUESTION: Look at the question closely - do they want you to draw a linear or a circular restriction map? It is good to take note of the length of the plasmid, as well as how many cuts each restriction enzyme makes. I usually use the restriction enzyme that cuts the plasmid only once as a reference point, and figure out the rest by looking at common fragment lengths and some math. QUESTION TYPE: Usually a follow up to this, they ll do PCR to a particular section of the plasmid, and run some gel electrophoresis to the product, and ask you to explain the results. ANSWERING THE QUESTION: Is the same is how you would answer for confirmation, but if they give you weird bands that are longer or shorter than the supposed length --> Longer: Amplification of non-target gene, primers may have annealed to other regions Shorter: Partial amplification of sequence during PCR/ (deletion of bases) Page 5 of 8

6 Bacterial Transformation & CONFIRMATION QUESTION TYPE: They ll give you some -random plasmid- and some DNA insert that they want to insert into the plasmid, and transform bacteria with. The plasmid will usually contain some selectable markers (such as pglo or lacz) and antibiotic resistance. The demands of the question is that they want you to write down some flow chart showing the steps as to how you will clone the DNA insert into the plasmid and transform the bacteria (till the preliminary identification step) APPROACHING THE QUESTION: The question can be worth up to 12 marks so do it systematically! (1) First of all, look at the question: Does it want you to describe, or describe and explain? The latter will require a lengthier answer yeah :) (2) Look at the plasmid and the DNA insert - what selectable markers does it have? Where is the cleavage site? For which restriction enzyme does the DNA insert have a recognition site for? If the question asks to describe the steps: Do a restriction digestion of (plasmid name) and DNA insert with (restriction enzyme name). If question asks to describe and explain the steps: Do a restriction digestion of (plasmid name) and DNA insert with (restriction enzyme name). If the question gives other restriction enzymes that the DNA insert recognizes also (look at the two ends of the DNA insert, sometimes they give extra restriction enzymes), you need to explain why the other restriction enzyme is not suitable, e.g.: (Restriction enzyme name) is not suitable as it allows the insertion of the insert into Ori, an essential region of the plasmid. Ligate (restriction enzyme name) digested (plasmid name) and (restriction enzyme name) digested DNA insert with ligase to form recombinant plasmid vector. Transform competent bacterial host cells that are (antibiotic names) sensitive with the recombinant plasmid. Transform competent bacterial host cells with the recombinant plasmid. The bacterial host cells are (antibiotic name) sensitive to differentiate between transformants and non-transformants, and (antibiotic name) sensitive to differentiate between transformants with recombinant plasmids and transformants with non-recombinant plasmids. *** Usually, the antibiotic resistance sequence already present on the original plasmid itself is used to differentiate between non-transformants and transformants. The antibiotic resistance present on the DNA insert is used to differentiate between transformants with recombinant plasmids and transformants with non-recombinant plasmids. But still, READ THE QUESTION PROPERLY. Transformation is done using treatment with calcium chloride solution to prep bacterial hosts to receive recombinant plasmids, then a brief heat shock to create transient pores in the host membrane, and lastly, placed on ice to close a pores and keep the plasmid in the bacterial host. Select the transformed bacteria with recombinant plasmid on LB selection medium containing (antibiotics, X-gal if lacz gene is present) Page 6 of 8

7 Colonies that grow on the selection medium are likely to carry the DNA insert, because transformants would become (antibiotic name) resistant due to the insert and the (antibiotic resistant gene name) on (plasmid name). If lacz is selectable marker: (EXAMPLE) Insertion of the DNA insert would disrupt the lacz gene, hence beta-galactosidase would not be produced and recombinant bacterial cells would not be able to cleave X-gal. (Also, mention the colour of the colony as white!) If pglo is selectable marker: (EXAMPLE) Insertion of the DNA insert would disrupt the pglo gene, hence recombinant bacterial cells would not be able to glow. (Mention something about the colony s ability to glow) ***DNA insert might carry these selectable markers! so you might need to reverse what is written in the above. Read the question really really carefully, do not throw away marks unnecessarily! QUESTION TYPE (FOLLOW UP - CONFIRMATION): They will probably ask you describe and explain how you would confirm if the transformed bacterial host cells are indeed carrying the recombinant plasmids using a particular method (e.g. PCR or Restriction Digestion, they will never ask you DNA sequencing!). ANSWERING THE QUESTION: Extract plasmid DNA from the bacterial host cells. If restriction digestion: Do a restriction digestion of vector DNA with (restriction enzyme name -- same restriction enzyme you used in the earlier question). If PCR: Run PCR of vector DNA using primers that bracket the DNA insert. Run agarose gel electrophoresis of the resultant products. If restriction digestion: If the transformed bacterial host cells are indeed carrying the recombinant plasmids, the result from gel electrophoresis would show 2 bands, with one denoting (original plasmid length) fragments and the other showing (DNA insert length) fragment, i.e. the DNA insert is present. However, if the bacterial host cells are not carrying the recombinant plasmids, the result of the gel electrophoresis would only show one band, denoting the presence of only (original plasmid length) fragments. This is because (restriction enzyme) will cleave recombinant plasmids at the 2 sites where the DNA insert was attached, hence resulting in 2 fragments being formed, whereas for non-recombinant plasmids, the restriction enzyme will cleave only the original plasmid at the recognition site, leaving only 1 fragment formed. If PCR: If the transformed bacterial host cells are indeed carrying the recombinant plasmids, the result from gel electrophoresis would show 1 band, showing (DNA insert length) fragments, and that the DNA insert is present. However, if the bacterial host cells are not carrying the recombinant plasmids, no result would be obtained. This is because the primers are unable to attach to the DNA insert as it is not present in the nonrecombinant plasmid, and hence PCR will not take place, and no product is produced; however, for recombinant plasmids, the primers will latch on and the DNA insert will be amplified, hence one (DNA insert length) fragment will be formed Page 7 of 8

8 TRANSGENESIS Definition: Transgenesis is concerned with the movement of genes from one species to another. QUESTION TYPE: List out methods used to create GMOs/explain one method. (list is shown below) Method Ballistic DNA injection Plasmid vectors (Agrobacterium tumefaciens ) What it essentially is Using a 0.22 rifle with the bullet (containing target gene) coated in tungsten or gold to shoot the plant cell/tissues, the change in a cell/organism is brought about by uptake and establishment of introduced DNA Agrobacterium tumefaciens infects plant through wound, and contains T-DNA in the its Ti plasmid. After infection, T-DNA will integrate into plant chromosomal DNA, hence, if we use recombinant Ti plasmids (with tumour forming genes removed, and gene of interest inserted into T-DNA along with antibiotic resistance marker), we can insert foreign genes into plant cells! There is a threat to human safety when using antibiotic resistance gene as the marker to identify transgenic organisms. When the transgenic plants are eaten, the genes for antibiotic resistance may pass from the plant to the E. coli found in the gut, making them resistant to antibiotics and cause hard to cure diseases. The fear of spreading the undesirable antibiotic resistance among non-target organisms is also a threat, these antibiotic resistance genes may then be passed on to other potentially harmful bacteria in the environment. GMOs are usually able to withstand environmental stress better than other organisms within the eco-system and they may affect the food chains in undesirable way if they are pests! Pronuclear injection An egg is selected from a female and it is fertilized artificially in a test-tube. The nucleus is injected with multiple copies of target gene via a glass micropipette or needle. The transformed egg is cultured in a petri dish into an embryo and implanted into the oviduct of a recipient (surrogate) mother to complete its development naturally. The process is inefficient: Only 2-3% of injected eggs give rise to transgenic animals and only a proportion of these animals express the added gene adequately Electroporation High voltage electrical impulses to create temporary holes in the membrane. Something like how we get bacteria to take up plasmids ;) If it s a plant cell, we need to create protoplasts first -- remove the cell wall. QUESTION TYPE: ESSAY QUESTION ON GMOs (see attached!) Page 8 of 8