Minutes 20101025 TA BIOL 208

From SnOwy - Ed's Wiki Notebook

TA BIOL 208

• What is a probe
• Types of probes
• Gene specific
• Labelling
• Stringency
• Restriction Mapping -- determine where the restriction sites are provided corresponding restriction enzymes
• The amount of a certain insert can be determined by the amount of cDNA present

Disambiguation

• Gene families -- use conserved regions -- may be exactly the same (DNA binding motif)

Answers

• M 134 - cDNA because we’re probing a protein
• Processive (Adjective): The property that an enzyme is firmly attached to a strand of DNA and will run to completion with high fidelity -- e.g.: Reverse transcriptase is not processive; it will often fall off before copying the entire mRNA

Hi Team

Here are some points that I will bring up at the meeting based on the readings assigned for the present tutorial.

Text (T): 117 - 134 / Manual (M): 121 - 142

• Verification Desired: T 118 - Is this correct: DNA polymerase can denature dsDNA.
• Terminology Learned: T 120 - Hybridization of imperfectly complemented DNA fragments -- called "Heteroduplex".
• Verification Desired: T 121 - "Hybridization" vs. "Reverse Hybridization":
  • "Hybridization": A filter has DNA fragments which are unidentified; a labelled probe with a known sequence is then used to bind with these fragments.
  • "Reverse Hybridization": A filter has DNA fragments which are known sequences; a labelled probe with a complex collection of unidentified sequences is then applied.
  • The intention of course is to identify the unidentified sequences given their complementation with the known sequences.
• Phenomenon Learned: T 123 - High stringency: High temperature + Low salt concentration (tends to denature).
• Terminology Learned: T 123 - "Heterologous Probe" instead of "Orthologous Probe" (chicken gene, human probe).
• Terminology Learned: T 124 - "Colony Lift" / "Plaque Lift" -- dish of probed clones are retrieved with nitrocellulose or nylon sheet
• Verification Desired: T 126 - Is this correct: "Back Translation" is a computational process, rather than a literal one which uses some molecular machinery.
• Terminology Learned: T 128 - "Guessmer" - a short nucleotide molecule composed of the guessed sequence given a back translation.
• Technical Question (Not to be discussed in a tutorial): T 128 - Does anyone use special nucleosides such as inosine for degenerate probing for each of the six frames (back translation)? I would suggest this can be accomplished if mapped to the Backpacking problem computationally.
• Clarification Learned: T 128 - "Expression" is with respect to protein detection, not transcriptional products.
• Verification Desired: T 130 - Is this correct: A "Secondary Antibody" has a label, we use it to bind the primary antibody; the primary antibody offers specificity for the target protein of interest while the secondary antibody can be identical so long as it binds with some identical region of the primary antibody; the whole reason for a secondary antibody is thus economic / efficiency.
• Terminology Learned: T 129 - "Linear Epitope" - a short contiguous peptide sequence which is recognized by a primary antibody regardless of the linear epitope's three dimensional conformation.
• Blank Desired: M 125 - Blank 2; is this correct: "Phosphoimaging Plates"?
• Blank Desired: M 126 - Blank 1; is this correct (Homologous Probe): Same organism, known / identified / sequenced gene; however, subsequence missing / unsequenced / unsure
• Blank Desired: M 126 - Blank 2; is this correct (Heterologous Probe): Different organism, orthologously derived probe
• Blank Desired: M 127 - Blank 1; is this correct (Gene Family): In a given genome for one organism, a gene family corresponds to a set of highly similar genes (by sequence identity) which occurs in a locus of a chromosome.
• Blank Desired: M 127 - Blank 2; is this correct (Gene Family: which parts of the genes therein are distinct?): In a given gene family, unless all of (1) the protein product and (2) the untranslated regions and (3) the introns are identical, the genes will be completely distinct.
• Notice: M 127 - Learning Task - skipped for now, will return later.
• Verification Desired: M 130 - Is this correct ("Filling ends of restriction enzyme sites"): -- this doesn't appear to be very useful in real life; appears to only fill in the few nucleotides which are complementary to a sticky end; is this conjecture correct?
• Why Desired: M 134 - Why; Is this correct (Use radioactively labelled probes in a hybridization reaction "preferably on cDNA libraries. Why?"): We do this to reduce the search space to only those sequences which are transcribed and translated; we do this because we do not expect to find / are not interested in finding a matching sequence in intergenic / untranslated / intronic region of the genome.
  • CORRECT ANSWER: These are protein sequences -- only cDNA is needed.

Weekly review questions

(1) I don't understand how the answer was derived for this. We want to produce a probe for the anti-sense strand ending at the 5' of the T3 site. Why aren't XbaI and SacI the best choices, as they do not include any of the muticloning site but *do* include the entire T7 site.

(4) Do we in practice in fact create all of the 29-mers to probe for the target sequence? Are there rational ways to suspect a particular synonymous codon? For instance, is there a known nucleotide distribution for the target organism, does that matter for this class?