Molecular biology

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Flashcards on Molecular biology, created by reynoldslaura on 04/30/2013.

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Created by reynoldslaura almost 6 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