I need to design an assay to test a gene that has a single polymorphism of C to T at one site. I was wondering if an OLA assay would be the best route to go and how would I start the design process.
217596
I also don't know what you're referring to. I've never heard of something called an oligonucleotide ligation assay. Usually I stop myself before jumping in to say that something is wrong just because I've never heard of it, because there is plenty of stuff I've never heard of. But let me tell you what those words mean to me. Oligo means few, so an oligonucleotide is a string of nucleotides, like a primer. It's generally used as either a primer or a probe. Ligation is sticking something together. In the same context as oligonucleotides, that would generally mean subcloning something.\n\nThere are a lot of ways to check sequence, but none of them really involve ligating oligonucleotides. I know two ways to check for this, one vastly preferable to the other. Get primers flanking the region, PCR amplify, and sequence. Really, you can stop reading there and just do that. The other assay is for genotyping that has to be done often, where it is too expensive to sequence everytime. Get primers flanking the region. There will be one forward primer, but two reverse primers. Each of the reverse primers will match one of the polymorphisms, but one of them will have a longer tail. Run the products on a gel and differentiate the two by size.\n\nIt's a pain to do and doesn't work well. Use the first method.\n\nNow I'm going to look up "OLA" and see if I can learn something new today.
498803
sorry im a high schooler who doesnt know exactly how to do it but i think to approach it:\n1) find genotype thats just a little bit of guess and check although you know which ones are dominate since they give capital letters and you know they're heterozygous\n2) order youll have to find again with logic and guess and check\n3) genetic distance will be the times that it screws up from what would theoretically happen. Each gene locus in distance is about 1% so the number of percents that accidently cross will be your distance of alleles in map units\n4) this sounds like you would have to do this inorder to get #3... I really dont know too much about this but thats my stab at process of how to solve\nIf there is a pro here, I'd like to know how to do this too
603911
Try a rough DNA isolation, some things u need are detergent, lemons, and a sample like onion.\n\nSimple diffusion experiments with onions, salt, water, etc..
369394
There are too many things that might have gone wrong.\nYou are saying your template is a genomic DNA and then that it is plasmid ("if I shouldn't use a plasmid DNA") ? It can't be both, unless you mean you have a genomic library in a plasmid...\n\nplasmid templates are very good for PCR so that is definitely not the problem.(Unless the plasmid preparation is not good and you have e.g some ethanol or isopropanol or whatever in it.)\n\nIf you meant that you have a choice between genomic and plasmid template  go for the plasmid \nYou have to check again the design of your primers, the putative Tm, the size of the DNA so that you let elongation occur for enough time, etc...\n\nSome polymerases cannot cope with very long and difficult templates under usual conditions so you might have play with the conditions or change polymerase or use a polymerase mix.\n\nWe don't have enough info to be more specific. You just have to go through the design again carefully to see if there are any mistakes and then start changing conditions (Temperature, Mg concentration, annealing temperature varying with cycles, polymerase, etc)\n\nAlso if you designed the primers based on the cDNA but the template has introns, did you check that there are no introns disturbing the sequence which the primers should be annealing to?
450998
click here:http://rds.yahoo.com/_ylt=A0geuuCMJFdFWDsB6.FXNyoA;_ylu=X3oDMTE2NXZmaDBwBGNvbG8DZQRsA1dTMQRwb3MDOQRzZWMDc3IEdnRpZANERlg1XzMw/SIG=122s68rjs/EXP=1163425292/**http%3a//www.wellcome.ac.uk/en/fourplus/DNA.html
46067
The PCR process consists of a series of twenty to thirty cycles. Each cycle consists of three steps \n\n The double-stranded DNA has to be heated to 94-96°C in order to separate the strands. This step is called denaturing; it breaks apart the hydrogen bonds that connect the two DNA strands. Prior to the first cycle, the DNA is often denatured for an extended time to ensure that both the template DNA and the primers have completely separated and are now single-strand only. Time: 1-2 minutes.\n\n\nAfter separating the DNA strands, the temperature is lowered so the primers can attach themselves to the single DNA strands. This step is called annealing. The temperature of this stage depends on the primers and is usually 5°C below their melting temperature (45-60°C). A wrong temperature during the annealing step can result in primers not binding to the template DNA at all, or binding at random. Time: 1-2 minutes.\n\nFinally, the DNA-Polymerase has to fill in the missing strands. It starts at the annealed primer and works its way along the DNA strand. This step is called extension. The extension temperature depends on the DNA-Polymerase. The time for this step depends both on the DNA-Polymerase itself and on the length of the DNA fragment to be amplified. As a rule-of-thumb, 1 minute per 1000bp.\n\n\nRestriction Fragment Length Polymorphism (RFLP) is a technique in which organisms may be differentiated by analysis of patterns derived from cleavage of their DNA. If two organisms differ in the distance between sites of cleavage of a particular restriction endonuclease, the length of the fragments produced will differ when the DNA is digested with a restriction enzyme. The similarity of the patterns generated can be used to differentiate species (and even strains) from one another.
314466
a punnett square is a genetic tool designed by reginald punnett that biologists continue to use in order to determine the probability of an offspring expressing a particular genotype.\n\ncertain facial features are dominant such as curly hair, free ear lobes and dimples. the curly hair trait will be used as an example: a homozygous curly-haired male with genotype CC, meaning he carries both dominant alleles and has curly hair, is crossed with a homozygous straight-haired female with genotype cc, meaning she carries both recessive alleles and has straight hair. typically, the dominant trait is denoted by capital letters and the recessive by small letters. \n\nhomozygous means that both alleles are the same, while heterozygous means both alleles are different. so, if one of them were heterozygous then its genotype would be Cc, meaning it has one gene for curly hair and the other for straight hair.\n\nusing the given genotypes, a table is made putting one of the genotypes on the top row and the other genotype down the left column. as a rule, the male's (m) genotype is usually written down the side of the square, while the female's (f) is listed on the top. we'll go down starting at the first column to put both genotype letters in the blank, remembering that in a genotype the dominant gene is written first. \n\nm&#92;f c  c\nC  Cc  Cc \nC  Cc  Cc\n----------\nresult: 4 Cc (curly hair)\n\nwhen the genotype paired up results in Cc, the dominant allele is shown in the phenotype, meaning the offsprings with genotype Bb will have curly hair. all of the offsprings from this cross demonstrate the phenotype for the dominant allele, though they are all heterozygous.\n\nnow let's try to cross a homozygous curly-haired male (CC)with a heterozygous curly-haired female (Cc).\n\nm&#92;f C  c\nC  CC  Cc\nC  CC  cc\n---------\nresult: 2 CC : 1 Cc : 1 cc\n3 C_ (curly hair) : 1 cc (straight hair)
74574
Ha Ha I just had an exam on the TCA.  \n\noxaloacetate is also used to make the Aspartate and Asparagine which are used to make Pyrimidines.  Oxaloacetate is also used with the enzyme PEP carboxylase to yield PEP (Phosphoenolpyruvate).  So if a cell is trying to make biosynthetic products (amino acids, nucleotides, heme, ect.) it may stop the TCA cycle and use its intermediates here instead.  \n\nAnother explanation\n\nMalate is oxidized (losses electrons) to form oxaloacetate (step  8 of TCA).  The electrons lost are added onto NAD+ to form NADH.  If there is a high concentration of NADH, there is normally a low concentration of NAD+ meaning that the malate stays as malate because it cannot lose its electrons to NAD+ to yield oxaloacetate.
503639
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=iga.section.962\nhttp://www.ndsu.edu/ndsu/abergstr/webcourses/genetics/tpt/tpt01.htm\n\nHope this helps!
36181
- with PCR you amplify a DNA sequence (like ATGGTAGCATGATC etc etc etc)\n- P = polymerase (thats the name of the enzyme that does the amplification\n- C = chain (there are actually two chains: first the chain of nucleotides ATGGAGACCCTAetc and second the chain of polymerization reactions)\n- R = the enzymatic amplification reaction\n- you can only do a PCR with a DNA double strand, because you need two starter oligonucleotides that bind to the initial template that you want to amplify; they resemble a short DNA sequence of the complementary (opposite) strand; they work best when they have a length between 18 and 20 nucleotides. When you do a PCR, you will most likely know the sequence of these oligonucleotides and you will order them from a company who designs them for you (you just give them the sequence you desire and they make them fo you)\n- so when you do a PCR, you have to offer the polymerase enzyme some material so that it can work: i.e. the template DNA that you want amplified, the starter oligonucleotides and especially the single nucleotides A, T, G, C\n\n- for RFLP you will need an enzyme that cuts the DNA strand\n- restrictions enzymes cut specific sites\n- they have names like BamHI EcoRI etc etc etc\n- example: BamHI cuts at the site GGATCC and only there\n- so when you have a DNA sequence that is, say 2000 basepairs long, and you have only one GGATCC, the enzyme will cut only once. If the DNA sequence was linear (i.e. it has two ends like a sausage) then you will get 2 DNA fragments. If your DNA is circular, you will get one fragment\n- so think in larger dimensions: if you cut a tremendously long DNA sequnece, you will have more GGATCC sites, consequently you will get more fragments.\n- so if you know your sequence, you can just do your simple math calculations and predict the fragments and their lengths: from the fist GGATCC to the second its, for example 10,010 basepairs, from the second to the third xxx and so on\n\n- R = restriction (cutting the DNA strand into pieces)\n- F = fragment (the pieces of DNA that arise when cutting the large one)\n- L = length (the different lentgs of DNA pieces that arise during the cutting)\n- P = polymorphism (referring to the fragment pattern when comparing them to the pattern of different samples)\n- the actual RFLP comparisons are made in the gel, because sometimes you don't have the exact DNA sequence of every DNA you want to analyze\n- for example, you cut the genomic DNA of two or more related bacterial species. And lets say you will know the exact sequence of only one of them. For that one you can make predictions about fragment lengths based on your calculations. For the others you can't, because maybe they have mutations in their sequences.\n- so when you compare the fragments to each other and to the one you initially precalculated, you might find that the other related species reveal different fragments. That means the sequences af the repated species have alterations in the site where the restriction enzyme was supposed to cut.\n- So the interpretation will be that specific DNA sites have an altered sequence to the sequence that was given to you\n- you can also make predictions about total mutations in the DNA, if you know how long the total DNA was and if you know how many restrictions sites there are and how many were not cut with a specific enzyme
381917
This is a shot in the dark. It sounds to me like gene dosage is the number of active copies of a gene in an organism. I'm assuming you're talking about something like E. coli engineered to produce insulin, so if each bacterium has one copy of the insulin gene inserted into its genome, that's one dose. If the bacterium has 100 copies of the gene, it can produce 100 times as much insulin. Just my guess.
295140
Yes, you can use it for over expression.  Make sure your plasmid has the proper self cloning sequences, one plasmid per cell won't do you much good.  \n\nPrecautions?  \n\nMake sure your gene is pure, as in not having anything that might ruin your experiment. Test culture your target bacterium first to weed out the non functionals and then cultivate the good ones, the ones that produce your targe protein.  You might need to assay for this stuff though, unless there's one of the specialized agars that will detect your target protein.
15253
Alleles are different 'forms' of the same gene.\n\nFor example, there are three alleles of the gene for blood groups; alleles A, B and O.\n\nIn diploid organisms like ourselves we have two complete sets of genes, so there are various combinations of alleles. One allele is inherited from the mother and one from the father.\nAA - blood group A\nAO - blood group A (because A is dominant to O)\nBB - group B\nBO - group B (B dominant to O)\nAB - group AB (alleles are codominant - equal dominance)\nOO - group O\n\nGeneologically, we can follow the inheritance of various characteristics (determined by alleles) and similarly blood groups can be used to eliminate or confirm parentage.\n\nThe 'classic' geneological example is the family tree of Queen Victoria of Great Britain. This follows the inheritance of the sex-linked gene for haemophilia.
138792
You're trying to say you have a synthetic chromosome. I would check all the experiments for contamination, and check the cells for endogenous expression before jumping to that conclusion. There are a lot of hurdles to leap, as mentioned in the other posts.
439214
From Gene to Protein\nScientists that worked on Protein Synthesis:\n1. Archibald Garrod: First proposed the relationship between genes and proteins.\n2. George Beadle and Edward Tatum: working with Neurospora , formulated the one-gene-one-protein hypothesis. This was later changed to one-gene-one-polypeptide. Since some proteins are produced from more than one gene.\n\nThe Problem: Since there are 20 different amino acids; and these amino acids are used to make proteins, how does the information get from the DNA to the final protein product? In a DNA molecule the only variable part is the 4 different bases. How can 4 bases, or combinations there of , code for the 20 different amino acids?\n\n1. If 1 base coded for 1 amino acid = 41 (4) amino acids coded for.\n\n2. If 2 bases coded for 1 amino acid = 42 (16) amino acids coded for.\n\n3. If 3 bases coded for 1 amino acid = 43 ( 64) amino acids coded for.\n\nNotice the large gap between 2 and 3. It was concluded that each coding part of DNA (codon) consisted of 3 bases. Since 64 is larger than the 20 amino acids, it stands to reason that some amino acids are coded for by different codons. Why is this important?\n\nTranscription: DNA is too large to leave the nucleus. If the program it contains must leave the nucleus, an intermediate must be produced. This intermediate is called m-RNA. m-RNA is formed in the following way, RNA polymerase binds to, a region of the DNA called, the promoter. Here is found the start codon TAC which codes for the amino acid methionine. Once this has been established the m-RNA sequence is produced growing from the 5' - 3' region. This occurs at the rate of 60 nucleotides / sec. The terminator sequence will keep the process from going on indefinitely. The eukaryotic m-RNA must be modified before going to the ribosome, while the prokaryotic does not.\n\nTranslation: t-RNA : a specialized form of RNA used to carry a specific amino acid to the ribosome and place it in the proper position. t-RNA contains 80 nucleotides in the form of a clover leaf. The t-RNA must attach the correct amino acid to itself. The process is as follows:\n\n1. The specific amino acid reacts with a specific enzyme and ATP to release 2 P and combine the AMP and the amino acid.\n\n2. The t-RNA will replace the AMP and bond with the amino acid.\n\nRibosomes: Before translation ribosomes consist of 2 separate sub-units. As translation proceeds the ribosome parts come together to allow protein synthesis to begin.\n\nThere are 3 steps to protein synthesis:\n\n1. Initiation: m-RNA binds to the small subunit of the ribosome. There is 1 binding site for the m-RNA, and 2 binding sites for the t-RNA. The P site holds for the t-RNA carrying the growing polypeptide and the A site for the t-RNA carrying the next amino acid. AUG ( start codon ) is in place and the appropriate t-RNA delivers the amino acid Methionine. The large sub unit then joins and creates a functional ribosome.\n\n2. Elongation: The chain moves from the 5' - 3' direction caused by the use of GTP and several protein-enzyme complexes. As the polypeptide chain is produced the t-RNA move from site A to site P, and then are released into the cytoplasm. The polypeptide chain continues to grow, and is held to the ribosome by the t-RNA at the P site.\n\n3. Termination: The termination codons: UAA,UAG, and UGA stop the process by adding water to the end of the chain instead of an amino acid. In order for the protein to become functional, it must be folded into its appropriate shape,and/ or have some amino acids removed, and/or have some of the polypeptides modified by adding sugars or phosphate groups to them.\n\nThe Wobble Phenomenon: There are only 40 different types of t-RNA and 64 codons. This means that some of the t-RNA can pair up with several different codons. This can occur because the third base of a t-RNA molecule can form a hydrogen bond with more than one kind of base. U in the third position can bond with A
134064
(m)\n\nmetabolic pathway analysis, it should be considered that many enzymes operate with low specificity (e.g. nucleoside diphosphokinase, uridine kinase, transketolase, aldolase), so that various substrates and products can be converted. Here, we analyze the effect of enzymes with low substrate specificity on the elementary flux modes (pathways). We also study the benefits of two different approaches to describing multifunctional enzymes. The usual description is in terms of (overall) enzymatic reactions. At a more detailed level, the reaction steps (half-reactions, hemi-reactions) of the formation and conversion of enzyme-substrate complexes are considered. Multifunctional enzymes operate according to various mechanisms. This is illustrated here by the reaction schemes for the different enzyme mechanisms of bifunctional enzymes. For enzymes with two or more functions, it is important to consider only linearly independent functions, because otherwise cyclic elementary modes would occur which do not perform any net transformation. However, the choice of linearly independent functions is not a priori unique. We give a method for making this choice unique by considering the extreme pathways of the hemi-reactions system. A formal application of the algorithm for computing elementary flux modes (pathways) yields the result that the number of such modes sometimes depend on the level of description if some reactions are reversible and the products of the multifunctional enzymes are external metabolites or some multifunctional enzymes partly share the same metabolites. However, this problem can be solved by appropriate interpretation of the definition of elementary modes and the correct choice of independent functions of multifunctional enzymes. The analysis is illustrated by a biochemical example taken from nucleotide metabolism, comparing the two ways of description for nucleoside diphosphokinase and adenylate kinase, and by several smaller examples.
249729
Many of the orignal studies were based on mutations in bacteria which halt growth or have other phenotypes, where it was found that mutations could be labeled into several groups, and that if bacteria from different groups mated, then the phenotype is restored.  From this it was deduced that the mutations were in coding regions for proteins that exist in a network ie A to B to C, and usually proteins like A and B were in a complex.  Today its a large part of cell and molecular biology to figure out if proteins are in complexes, and what the complex does.  Its not necessarily important that A and B are in a complex, but what that complex does; since that is the proteni function in cells.  So if A and C are in a complex, the complex has a certain function.  if B and C are in a complex, then there is a different function.  Drug companies have been exploiting this type of knowledge for a long time, but what is more impt is the understanding of how proteins interact with other proteins, and how they regulate cellular functions.  Once this is understood, then drugs can be rationaly designed.  Even then, however, it's not possible to guarantee patient efficacy.  \n\nWe just had a great lecture by Gordon Mills, an expert in the PTEN protein pathway, and he talked about the dozens of drugs made against various members of this pathway, and noted that so far none of these drugs are effective in patients, although they work in the test tube and in mice.  So, drug discovery is a very difficult issue and takes billions of dollars for one drug to be delivered to market (and 10 years or more).