MedGen

Thanks Jimmy, but I am aware of the potential of certain haplotypes predisposing to disease within certain populations. I want to see if these extend beyond what is normally detectable by using standard measures of linkage disequilibrium. What I was looking for was more the technical aspects of phylogenetics. I wondered whether it was possible to infer intra-population phylogenies from the haplotypes present in a population. The extensive LD across the MHC doesn't really lend itself to well to this idea unfortunately. I was thinking of testing the idea on a slightly less extensive LD rich area to start with.

Synthesis is always in the 5' to 3' direction. The key to is to remember that the strands are anti-parallel. The actual direction of transcription whether on the + or - strand depends on the position of the promoter elements. The strand that will contain the same sequence as the mRNA is known as the sense strand, and the one that is actively transcribed from is the anti-sense strand. Here's a little representation using your example: 5'-AGTGGGCCG-3' - sense strand + 3'-TCACCCGGC-5' - anti-sense strand - 3'-TCACCCGGC-5' - anti-sense strand 5'-AGUGGGCCG>>>>>>3' - mRNA If the gene was transcribed in the opposite orientation: 5'-AGTGGGCCG-3'[/b] - anti-sense strand + 3'-TCACCCGGC-5' - sense strand - 5'-AGTGGGCCG-3' 3'<<<<<UCACCCGGC-5' - mRNA Note that the denotion of + or - remains the same. So the orientation of the gene itself is denoted by whether it appears on the + strand or - strand. This is for the purpose of having a constant reference when mapping genes to a specific locus and aligning sequences, particularly with respect to transcriptional direction and retrotransposed elements, but that's another story. Hope that helps answer your question.

Firstly I will add a caveat that I am not entirely familiar with the exact mathematical models and algorithms involved in this (phylogenetics), so I might not be able to clear up any misunderstandings. I'm trying to see if there are representative haplotypic structures inherent to particular populations beyond that which is initially apparent by local LD; lets call them extended haplotypes. Now I want to see if there is any evidence for these extended haplotypic structures (background haplotypic structure) that may confound the association signals seen in GWAS due to either a) insufficient marker coverage on commercially available SNP chips, or b) whether fallacious assumptions about (supposedly) genetically isolated populations are representative of their purported ancestral population. I hypothesise that by looking at the divergence of these specific population haplotypes using phylogenetic inferences (based on an ancestral haplotype) it will be possible to determine if association signals are genuine for their associated disease, or whether they are an as yet un-accounted for confounding issue. This is of particular note where (perhaps to be expected) certain genetic variants are only associated with specific populations. So, to my question, are there specific parameters that need to be applied, or variables that need to be accounted for when deriving phylogenies of haplotypes? Thanks in advance.

I'm not familiar with chemiluminescence visualisation, but that looks like it might be a problem with the camera rather than the gel or the assay. I say that because it looks like it is overlaid on the gel, you can still see through it where the band smears are. What system are you using to visualise the gel with? i.e. BioRad Geldoc, etc.

Whilst I agree with that to a certain extent I've found that lectures can only deliver a basal level of knowledge, the skeleton as it were. What is useful about them is the ability to ask questions and tangible examples. But then again, not all students read up on the lecture before they go to it.

If you're trying to extract hydrophobic photosynthetic pigments then you need to employ a hydrophobic solvent. I'm guessing you'll be using chromatography, thin-layer perchance?

Well done on the promotions.

profeshcer: DNA dependent RNA-polymerase catalyses mRNA production from a DNA template, RNA dependent DNA polymerase initiates DNA replication by priming with RNA which is later degraded and replaced by DNA. DNA synthesis itself (replication) is mostly an RNA-free affair, with the exception I've noted. So if an antimicrobial compound inhibits DNA synthesis then it prevents the cells replicating. If it inhibits RNA synthesis then it prevents transcription and thus gene expression. Importantly these are two distinct mechanisms and it is worth noting that. This isn't an issue for debate, it is whether or not rifamycin inhibits replication or transcription, and your current inability to differentiate between the two. NB Note that a medical handbook will generally gloss over the specific mechanisms of drug action, but you are still mistaken. edit: crap, missed CharonY's post above

I also forgot to mention processed pseudogenes also arise by reverse transcription.

My bold, this is a nit-pick, and I'm sure you didn't mean it, but LINE-1 mean anything to you? Not a reverse transcription isn't a virus only affair, unless you count transposable elements as virii because of their evolutionary past.

Do you mean once a protein is translated and when ATP is synthesised?

MHC typing is effectively characterising the relevant genotype or phenotype of a patients MHC at 6p21. It's very important for a) understanding anything about the immunology of an individual b) matching donor and recipient for organ/tissue transplants. Not to mention the applications in medical research for understanding the genetics and molecular mechanisms of autoimmunity. RFLP's are the simplest by far, the alternative (and one the we'll no doubt see as clinically routine in the not-too-distant future) is to sequence that region of the MHC and genotype them that way. Alternatively you can interrogate the region with SNP chips and pick out the individual genotypes that are relevant. I'm not sure how they seriologically type HLA alleles, but I'm guessing its done with a) standard histology or b)immunohistochemical staining, c) in vitro assay. The HLA locus is probably one of the most important genomic regions as far as an immunologist is concerned.

Evolution is effectively just the changing frequency of alleles in a gene pool over time. What forces drive that change is another matter, whether it be a stochastic process of genetic drift or direct constraining negative selection is another matter. People often think humans are some sort of pinnacle of evolution, or that we are no longer evolving. We split onto the homo genus lineage from our LCA with chimpanzees some 5-10 million years ago, anatomically modern homo sapiens has probably only been around for last 200,000 years, bugger all in the grand scheme of things. It is also worth noting that we are still evolving, particularly at the genetic level, lots of funky stuff going on there in terms of variation. The only things that have changed are the types of selection pressure.

That's a very high GC content and Tm. Its always worth using a primer calculator when designing primers. I personally use the nearest-neighbour Tm as a guide for annealing temps. You want a GC% around 50%, just because it simplifies the reaction. High GC content 60+ often requires a specifically optimised reaction buffer. I always add one extra reaction when making up a master mix, it allows for a larger margin or error when pipetting between several different tubes. If you only use one primer then you will only get linear amplification, you'll need a reverse primer for exponential amplification. Its worth checking with the supplier of the PCR kit (website and protocol supplied) for optimum reaction conditions and reagent concentrations. Generally speaking I work with primers at 100µM each and dNTP's at 10mM. That usually means that most of the reagents get used up but don't become rate limiting in later cycles. Standardise your reaction volume, say 20, 25 or 50µl. That way it makes it easier to calculate concentrations and hence the required volume. Always calculate the water requirements last as this effectively just provides a medium for the reaction to occur in. Calculate what you need for one reaction, then multiply up to the number of reactions you are carrying out, it makes the whole process easier. Work on the basis of the recommended thermal profile supplied by the manufacturer, they are optimised for that enzyme. To get the annealing temp you'll need to run an annealing temperature gradient, it will be a function you can programme on your thermal cycler. Your steps seem long. Generally the initial denaturing last for no more than a minute, depending on the extraction product size and target sequence length. The same applies to the annealing time, only needs to be around 5-30 secs. The extension time depends on the length of your intended amplicon. The longer it is the longer you need to run the extension time. The protocol provided with your enzyme should give a guide of the processivity, generally stated as bp(or kb)/s. e.g. you need a product of 4kb and the processivity is 1kb/30s, run the extension for 2 mins and then a little more, say 5-10 secs. The final extension time is usually 5-10 mins just to finish off the final cycle so all the sequences are the right length. Bare in mind when setting up the thermal profile that although the polymerase is thermostable, it doesn't mean it is indestructible, it still needs to be stored in -20 freezer, and on ice when being used. Additionally the repeated thermal cycling will denature the enzyme slowly. The annealing temp gradient needs to be designed on the basis of the nearest-neighbour Tm. I generally go up and down by 6 degree with the calculated Tm in the middle. e.g. my Tm is 59oC, so I'll set up a temp gradient of 53oC to 65oC. If you have a hot-start enzyme then start the reaction with a hot-start, it will increase the specificity of the reaction and prevent unwanted non-specific amplification Hope that helps resolve your problems. Also when designing primers always have your target sequence in front of you, and if you want to sequence a particular region then you need to shift your primers up/downstream accordingly to account for interference from the dyes.

The 36-38 also refers to the general calculation that one molecule of glucose yields 36-38 molecules of ATP post-glycolysis and TCA.

How about looking in a textbook. <tag for homework?>

I wouldn't say the actual science involves politics (not counting funding here), but more the application of scientific discoveries on a large scale. For instance if a biomarker and test were developed that could detect cancers (any type) at the neoplasia stage with 99% efficiency, but cost £8000 ($12000) per person to implement, would the government subsidise or pay for it?

Have you tried looking in a biochemistry textbook? It often goes by other names such as Kreb's cycle or the citric acid cycle. Any biology textbook should have an overview of the pathway. Any thing more specific you wanted to know about the actual process?

Following on from Dak with the basic lipid membrane enclosed replicator; it also depends on what the replicator is. If it's RNA then it can form secondary structures that can catalyse reactions like an enzyme, called ribozymes. So you've got replicating molecules that can catalyse reactions, including their own synthesis, et voila!

In all fairness there have been some very important advances in recent years in genetics, mostly through the development of genome-wide SNP chips that allow genotyping at 500,000 SNP's across the whole genome. From there genome-wide association studies have been carried out to start probing common alleles in case-control studies. Using this GWAS approach there have been numerous risk association alleles attributed to a number of diseases, including autoimmune diseases. The barrier with this approach is the need for very high statistical power to detect the small effects that these risk alleles confer. The next step after determining the association is the functional study approach, which is underway on many of these susceptibility loci. Additionally you have the spectrum of variation across the genome that is being elucidated. The impact of copy number variation on the outcome and susceptibility to certain diseases is being investigated. A lot of this work isn't reaching the publics ears, its all rather complex, but I'm still a little surprised that even educated laymen haven't encountered this.

Not a lot usually, slight chance of reduced intelligence, but that's about it really. Bare in mind that the Y chromosome is still mostly silenced regardless. There may be some gene dosage issues since there are two SRY genes, but I don't think that's anything special compared to other structural variants and CNV's.

The key to it is considering the position of the hydrophobic aa's and the quaternary structure, if that helps at all.

Thanks for the correction.

IIRC allosterically is an indirect method of regulation, e.g. tyrosine/serine/threonine phosphorylation as seen in most signalling pathways that require the activation or inactivation of a protein, that requires the modification of the target protein. Inhibition mostly relates to the direct competitive inhibitors that compete with an enzymes substrate(s) in the active site thus preventing the enzyme from catalysing the reaction it would normally. Hence allosteric inhibitors do it from behind, they don't actively compete for the active site.