62921
Description
Flashcards by reynoldslaura, updated more than 1 year ago
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Created by reynoldslaura
over 11 years ago
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| Question | Answer |
| What are the requirements for performing the PCR reaction? | Template DNA, deoxynucleotide triphosphates, a heat stable DNA polymerase (e.g. taq DNA polymerase) and primers |
| What is the difference between northern and southern blotting? | Northern blotting is used to fractionate RNA and southern blotting is used for DNA |
| Give an example where you might use the northern blotting technique | When analysing the expression of follicle stimulating hormone-β in different tissue types |
| Why is PCR the preferred molecular diagnostic method of choice? | It is cheaper, faster and more sensitive than southern blotting (although southern blotting is still used for diagnosing some disorders e.g. Fragile X syndrome) |
| After 30 cycles of PCR, how many times will the target fragment be amplified? | Approximately 2³⁰ fold (as after n cycles of PCR there will be approximately 2^n copies of the fragment) |
| What 3 reactions are constantly repeated in the elongation cycle? | Binding of aminoacyl-tRNA (needs GTP), the peptidyl transferase reaction and translocation (movement of the ribosome one codon along the mRNA-also needs GTP) |
| What is the name of the initiating amino acid in prokaryote translation? | N-formyl-methonine (instead of methonine in eukaryotes) |
| How many ATP molecules are used for each amino acid incorporated into the protein? | 3x ATP |
| How many molecules of GTP are used during the addition of one amino acid to the peptide chain? | 6 x GTP (one molecule for everytime an aminoacyl tRNA binds to the ribosome and one for everytime the ribosome moves one codon along the mRNA) |
| In eukaryotes, what delivers aminoacyl tRNAs to the ribosome? | EF-1a |
| Name two groups of drugs active against prokaryotic ribosomes. | Any two from; Aminoglycosides, macrolides, tetracyclines and chloramphenicol |
| Name two drugs or toxins active against eukaryotic ribosomes | Ricin and diptheria toxin |
| What are restriction enzymes? | They are enzymes that recognise and cleave DNA at specific sequences. These are usually 4-6 base-pairs long |
| What does palindromic mean in terms of restriction enzyme cutting sites? | Both the top and the bottom strands have the same sequence |
| Give an example of a restriction enzyme that cleaves to give blunt ends | Hae III |
| Give an example of a restriction enzyme that cleaves DNA to give sticky ends | Eco RI |
| What is the average size of a DNA fragment generated by Eco RI digestion? | 4,096 bp |
| How many different fragments of DNA are generated if human DNA is digested with Eco RI? | 750,000 fragments (because the human genome contains 3x10⁹ base pairs of DNA and there is a recognition site for Eco RI every 4,096 bp therefore 3x10⁹ ÷4096 = 750,000) |
| What material is used to make the gel for gel electrophoresis? | Agarose or polyacrylamide |
| In southern blotting, why do we treat the gel with sodium hydroxide? | To denature the DNA- it breaks down the hydrogen bonds between the strands and allows us to "blot" DNA onto the nylon membrane |
| Give an example of a genetic mutation that can be identified via southern blotting | Sickle cell disease- it is a recessive genetic disorder caused by a single mutation in the β-globin gene. Can detect the mutation via the restriction enzyme Mst II |
| What base sequence is the initiator codon? | AUG- codes for methionine |
| Give two examples of stop codons. | Any two from... UGA, UAA and UAG |
| The genetic code is generally universal, however there are some minor variations. Give an example of where these may be seen. | In some mycoplasma (primitive bacteria) or in the mitochondria |
| How big are tRNAs? | 75-95 nucleotides long |
| What is the effect of a nonsense mutation? | A nonsense mutation will code for a stop codon therefore the production of that protein will be stopped prematurely normally leading to the production of an unfunctional protein |
| What occurs to the DNA sequence in a frameshift mutation? | There is a deletion of one or several nucleotides changing the way the codons are read. It may change all the codons downstream to the deletion |
| Name two unusual bases tRNA's may contain? | Any two from; Thymine, dihydrouracil, pseudouracil and methylguanine |
| What sequence do all tRNA's have at their 3' end? | CCA |
| What is an aminoacyl tRNA? | A tRNA that is attached to its amino acid |
| What are the 5 arms of a tRNA molecule? | They are the amino acid arm, the T arm, the D arm and the anticodon arm. (The variable arm may or may not be present) |
| What are the enzymes called that link tRNA's to amino acids called? | Aminoacyl-tRNA synthetases |
| Once the pre-initiator complex has been formed, what activates RNAP II? | TFIIE and TFIIH |
| What phosphorylates RNAPII? | TFIIH kinase |
| What phosphorylates Ser-5? | TFIIH, this triggers the release of RNAPII from the promoter |
| What does chromatin packing determine? | Whether or not DNA is accessible for transcription |
| What are the two types of heterochromatin? | Constitutive and facultative |
| What type of chromatin is permanently silenced? | Constitutive heterochromatin |
| What is the function of TBP (TATA binding protein)? | It binds at the TATA box and bends the DNA more than 80 degrees |
| What do TATA-driven promoters contain? | An initiator sequence (inr) and a TATA box |
| What do TATA-driven promoters contain? | An initiator sequence (inr) and a TATA box |
| What do additional upstream control elements like the CAAT and GC box do to the promoter? | They improve its efficiency |
| What are the two subunits of TFIID | TBP (TATA binding protein) and TAF (TBP associated factors) |
| What does the binding of TFIID to DNA initiate to form? | The pre-initiation complex |
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