Genotyping - Science method

To get a better chance of success, make sure to design primers so the SNP is at least 50 (prefer 100) nucleotides away from the primer site. That way you can avoid the messy start & end of the sequence.

If you have a sequencing core on site, then you could get the data by the next day.

I would start by diluting a new primer stock in TE (not water). Set up a pcr with positive control using normal amounts of Mg and primer and also tubes with 2x as much Mg and separately 2x as much primer. Before using all ragents thaw eack reagent and them mix by flicking the reagent tube. Sometimes when thawing frozen materials you get water layer on top and concentrated salt/oligo /protein layer at the bottom and you can pipette out water if you take up the upper layer of reagent. Be sure to dilute your primer in TE in case the water has nucleases in it which chews up the primer. run the samples with a dna ladder to check that this is not a gel problem...if you can see the ladder all is well with the gel

To enhance the efficiency of your genotyping workflow, consider adopting automated technologies that streamline various stages of the process. Automated DNA extractors, such as the Qiagen BioSprint and KingFisher Flex, can significantly reduce manual handling by automating DNA extraction and can be seamlessly integrated with automated PCR setup systems. For a more comprehensive solution, the Fluidigm Biomark HD System offers an integrated approach for high-throughput PCR amplification and analysis, minimizing the need for multiple instruments. Additionally, capillary electrophoresis systems like the Applied Biosystems SeqStudio and the QIAxcel Advanced System automate the electrophoresis and imaging steps, drastically cutting down processing time. For even greater efficiency, consider utilizing Digital Droplet PCR technology, which provides precise, absolute quantification and rare allele detection, thereby eliminating the need for post-PCR electrophoresis. Adopting these advanced technologies could significantly expedite your genotyping operations, allowing for quicker and more reliable results.

I don't measure activity in units making Taq, what I do is preform a test PCR reaction using several dilutions of the enzyme in order to find the one giving the best amplification. In most cases dilution of 20-50X was the one that worked.

Hi Dayun Lee

choose an annealing emperature that does amplify and run a pcr set with 0. 1%, 2% up to 8%dmso and at some dmso concentration the pcr will probably be a clean band. If your gene has pseudogenes or is one of a family of similar genes then touchdown pcr or new primers might be needed.. You can perform primer gradient PCR to optimize the concentration of primers. Also, You can use pico molar concentration of primers. or use different gradient pcr such as with annealing and denaturation etc.

Thank you so much i'll look into it

When I say the primer is annealing in 2 places I had in mind the following. If the amplimer is 500 bases then the forward primer might be 1-20 and the reverse 500-480 but if the reverse primer can also anneal less well at 570-550 then you might get 2 bands on all amplimers. Increasing the annealing temperature may minimise the second band. It may be that your annealing temperature is just on the level that sometimes gives 2 bands and sometimes only 1. This really does not look like a gel running problem to me. Good luck

Paul

When crossing loxP mice with FLPe mice, the removal of LacZ and Neo leaves behind the loxP site. To genotype these mice, you can use the following methods:

As for determining heterozygosity or homozygosity:

Remember to validate your genotyping results and adjust primer design as needed.

I am attaching some articles for your reference-

https://www.nature.com/articles/ng0600_139.pdf

I think you should index the ref

One solution is to do a restriction digest of the genomic DNA, dilute the concentration and ligate to form circularized pieces of DNA. You can then use PCR to amplify outwards from the ends of your insert and sequence the product to identify where the insertion is in the genome. You can then design primers to amplify across the insertion locus to allow for genotyping.

Alternatively make several separate lines and sequence a large number of progeny from self fertilization and only keep lines that do not produce any offspring lacking the insert. After two generations you can be confident that it is not heterozygous.

Reading a bit late in the game, but I'm actively using "Hapl-o-mat" - it's pretty good, easy to use and open to a range of applications: https://github.com/DKMS/Hapl-o-Mat

Hi, Which mice do you have? If they don't have the additional tamoxifen-dependence then neurons also sometimes express the cre (probably early in development). Even the tamoxifen-dependent lines might have some 'leak'. You can find a lot of information if you have JAX lines on their website...Non-myeloid cell expression also might be dependent on the floxed construct i.e. some recombine more efficiently then others (or might even undergo spontaneous recombination with no cre at all).

If this is genotyping, it should be fairly straightforward.

Do you know your primers work?

If "no", then optimise your primers using clean gDNA of known genotypes and establish what sort of Cq values are associated with positives and negatives (for probes, negatives should be essentially "no amplification").

If "yes", then what sort of values do you typically see? Could it be that you are using

?

All of these things can be inhibitory/detrimental to PCR. Not enough target means you deal with stochastic effects (sometimes there's no target, sometimes there's some: high variability, high noise). Too much means you inhibit the reaction by saturation, and might also include excess contaminants that impair reaction efficiency. And dirty DNA is just...well, generally bad. Usually DNA is more forgiving than RNA>>cDNA, because genomic DNA extraction doesn't use quite as many horrible chemicals and solvents, but it can still be bad, and it might be worth checking just in case (nanodrop or equivalent).

Hi, glad to answer your queastion!

Here are some reliable and comprehensive sources of genotyping databases.

Shiwali Goyal

Hi,

Thanks so much again, I will try BLASTing and check that out.

At this size, primer dimer is very unlikely. Seems like your primers picked up something with the enzyme - 16S primers eg could easily do that. Can try to run less cycles and/or run a blast search on your primers

I guess much of the work is in loading all the gels? You could develop qPCR probes for your six genes. Then you could scratch most of the gels, save for the occasional quality control.

When you say the end base of the primer do you mean the 5' end or the 3' end ?

I would expect one missing base at the 5' end of a primer to make no difference but a missing mismatched base at the 3; end of the primer would be likely always to produce a good match of primer to template dna . producing a pcr product because there is a perfect match . If you have samples with known sequences for the presence amd absence of a snp you can use these to confirm the quality of your results but if all of your samples seem to contain the f1 allele then you probably have a problem

It looks like you can download the instruction manual but only if you have an IFU number which is on the box that the kit comes in

try

https://www.hain-lifescience.de/en/downloads.html and select your kit and enter the IFU number of the kit.

It could be due to bad DNA purification during extraction protocol. See line 3 and 5 where no apparent amplification band appears and it also occurs.

Yes you can.

Hi Michael Nicosia

Maybe the Vav-iCre strain (originally from the Kioussis lab) may help, pending on your experimental set-up.

For a characterization (of Cre expression) please see:

All the best & good luck with your experiment,

Michael

Thank you for your answers. I think I have to go with gDNA this time, since I've tried a couple of experiments with cDNA. It worked with my "synthesized gene" as control, but not very with my cDNA samples. I am working on animal samples. I'm good with my gene expression using the RNA extracted and cDNA, so there's not much of a problem wtih my quality and quantity. I'll move to gDNA as my next step. Thank you very much and I appreciate your help.

INclude a proper set of controls and try again.

I had issues with a left-border primer making a product in WT as well. We called it Lb1 (guessing it was the SALK LBb1) mentioned above. The higher annealing temperature strategy helped.

It could be that the dna is not pure and contains pcr inhibitors, Measure the od 260/280 which should be close to 1.8.Low valuse means the dna has a lot of protein so too little dna, Use 50-100ng of gna....too much dna can stop a pcr reaction. Try amplifying less dna which may dilute any pcr inhibitors. If these tries fail then lower the annealing temperature 3c in case this samplle has a polymorphism underneath on primer stopping the primer from binding, If that fails use 2mM Mg instead of 1,5mM MG in the pcr mix which can counteract pcr inhibitors. Looking at your gel the sample is working weakly so you will only need a small change to get a good strong amplification

There could be several reasons why you are not seeing a beta actin band in samples 11 and 12, while still observing the Cre band. Some possible explanations are:

It may be helpful to run the samples again to confirm the results and to check for any technical issues that could have affected the PCR. Additionally, it may be useful to try using a different set of beta actin primers or to optimize the PCR conditions for this gene specifically.

what about target-next generation sequencing? fast and cost efficient, but you need a nexseq 500 or ONT platform.

PCR is the most accurate method for molecular characterization.

Hi Lilian,

qPCR is used for measuring quantity of your target (ie, number copies of genes or precise sequences for qPCR on DNA, and expression of a gene for RT-qPCR on RNA or cDNA), whereas the castPCR will only used to genotype a variant, one mutation.

castPCR means "Competitive allele-specific TaqMan PCR", one evolution for the old but reliable ARMS-PCR.

see this paper for more explanations:

all the best

fred

Thank you, yes its a very expensive lesson...

One possible cause is dirty DNA. What is the OD260/280 ?. Try using the best dna sample ( od260/280 1.6-1.8) and run dilutions of this dna. Sometimes dna contains pcr inhibitors and diluted dna amplifies better with less dna but also less pcr inhibitor. If this fails then try using more Mg in your pcr mix.

Hello, Pushkal Sinduvadi Ramesh, Thank you for your valuable suggestions and the provided links.

Sounds like you have a mixed population of cells. Streak out to single colonies and try again.

Thanks for your help I have already found the primer

The Idea of genotyiping is to get to know if your gene of interest has exactly the same "letters" in both alleles (or both chains of DNA). If one letter (Or more) changes in one of the chains and the other stays the same it should be considered heterozigous. If both chains have some changes it sohuld be considered Mutated Homozygous. Or if it doesn´t change at all, it should be considered Wt.

You need both primers that can detect this changes. @

Dear Sonia Marina ,

often you have three or four primers depending on the stratgy of your geneotyping PCR approach.

For a three primer system one pair is binging in the WT-situation of your genotype resulting in a PCR product of i.e. 500 bp. When now a knock out was done, it's was traditionally usually with a homologous recombination and a neomycin resistance. You can find simplified information here:

When now, the knock out was successful the primer for the reverse primer is to far away to create a PCR product (due to the insert of the neomycin resistance) or the binding site is eliminated during reconbination.

Now, the thir primer is binding as reverse primer in the neomycin resistance and will create a PCR-product of different length i.e. 700 bp. In a heterozygous situation both bands are showing up.

The benefit over a system with two pairs of primers is, that you have an internal control for the PCR. You must get always a product. When you have a two pair system (where knock out and wild type are charaterized in individual PCR reactions). It may happen that you fail in one PCR suggesting a different genotype (i.e. knock out) while the truth is that it is a heterozygous situation and only your PCR failed.

kind regards

Soenke

In knock-out animal models, a homozygous (-/-) animal means both the alleles of the gene of interest have been removed (e.g by Cre dependent removal of loxP sites, thereby generating a flox animal for your gene of interest at a specific site), heterozygous (+/-) means only one allele have been removed by the same process, while a wild type has both of the alleles (usually functional).

To simplify, you need to breed the animals for a minimum of 2 generation to generate a homozygous (-/-) animal, while heterozygous (+/-) (not all of the offsprings though) can be obtained from 1st mating. In subsequent matings of heterozygous parents, homozygous offsprings can be generated and mating of homozygous parents would generate further homozygous ones.

RNA comprises the majority of the nucleic acids in a cell, which is why the samples without RNase treatment show a much higher concentration.

In theory, your genotyping reactions could still work since they will be using a DNA-dependent DNA polymerase, but you won't be able to accurately determine how much DNA you are loading into your reactions.

Looks like you have non-specific bands. And you are really over-loading your gel - try loading less PCR sample in the wells.

I second the vote to design some new primers. Primers are cheap, endless trouble-shooting is expensive.

Good luck!

Hi Sina

designing primers for qPCR follow the same rules as for classic PCR, at the exception that amplicon must have 100-150bp length and one of the primer need to be across splice, at most 3' end of the gene..

take a look at the UCSC database, there is a genome browser and typically you can choose to make available the track presenting primers that have been designed and published on the gene (do not forget the rules for qPCR primers). after designing or choosing, you can also test them by in silico PCR on the same site.

all the best

fred

Severine Roques did you ever get SPAGEDI to work on SNPS? I can't seem to find papers that did accomplish that although I also don't see why it shouldn't work...

And if yes, how did you make the SPAGEDI input file??

Hello Jessica Roy

Please go through one of my research which is attached below. The methodology explained in the paper is appropriate for the kind of situation you are in.

Hope this helps you!

Best!

Please read these papers, it may help.

Dear Suzanne,

If you are confident about your statistics, one or more assumptions behind the Hardy-Weinberg equilibrium must be false. It is most likely that your sampling of patients is not random and does not reflect the real population structure that they belong to.

There are two interesting possibilities.

First, their might be something unique about your group of patients which is linked to the alleles you have been investigating; i.e., the patients, because they all have the same type of medical condition may mean that the alleles you measured (which somehow underlies the medical condition), will be in disequilibrium. You could test this (perhaps) by looking at a second set of alleles that have nothing to do with the medical condition, and these we would expect to be in equilibrium.

Second, all of your patients may be from a population of people in which the alleles you are investigating are in disequilibrium (and the alleles are not directly linked to the medical condition they may all have). This might be the case if all of the patients were in a regional hospital serving an area which had little migration and mixing, but less likely if they were in a metropolitan hospital that serves a diverse city. You could test this by looking at other alleles not linked to the medical condition, and in this case we would expect them to be in disequilibrium too.

Regards, Andrew

As far I know, if you are sequencing, the most probable situation is that, or you know predicted sequences or you know sequences of the same genes in other model animals. So the positive control does not have sense to me in sequencing a gen. And the negative either. Different thing could be if you know the sequence and you would like to confirm or demonstrate the presence of the gen in different tissues. In this situation I would use specific primers and Real Time PCR, and positive and negative theoretical controls of tissues in whcho you have some data to assume thta the gen is there and not. But this is not sequencing.

Thank you for that clarification Tharaka D. Liyanage and at least it shows that there is no dna degradation in the gel running buffer.

The reason for diluting the first round pcr product is partly to minimise non specific bands and partly to dilute the first round primers to avoid getting more non specific primers and mainly to avoid huge over amplification. You have to remember that 10 cycles of pcr is 1000 times more product and 20 cycles is 1000000 times more product .You have run 35 cycles of first round prc then 30 rounds of second pcr .At these levels of amplification you can get so much pcr product that it self anneals and you get long smears of amplified product but no single specific band.

About running first round product it is usually because you only use nested pcr when there is almost no dna in the original sample so there is often nothing to see after the first round. If you could see it then possibly there would be no point in running the second round but cleaning up the amplification of the first round with higher annealing temperature or adding dmso might be good enough to get a good product. Remember that while you might be adding 50ng of dna in the first round pcr almost all of this is useless host dna and very little is Gondii infection dna so there are very few targets for specific amplification with your primers

Run an assay with 2 samples which have no dna in them and one normal dna sample.If the negative controls amplify bands of the right size then you have contamination

For the mutant gene to turn into the original gene, this is normal if the mutation is reversible. If a single nitrogen base changes, then a gene changes from a likeness to a pure bred, that is also a possibility, because if the gene is given the conditions to return to the original, it will return, just as the members of migratory birds return to their original homeland without guidance from the parents. The origin in the genetic material is the preservation of the organism and the surrounding environment that plays an important role in this conservation or change. The answer to your question: Is it possible for that to happen? Yes, because you don't know which one was the origin?

In simple words, the faster the ramp rate, the higher the chance for missannealing of your primers. Especially when they are long and have a high tm.

We had that situation a week ago for a complicated PCR. No additional ramp = several bands, ramp at 0,5°K/s 1 main band and serveral fade bands, ramp at 0,3°K/s only 1 main band.

I still want to know how to know the result from the excel sheet . whats parmeters should i look for

Use SYBR Safe DNA Gel stain (Life technologies S33102). This is less hazardous than Ethidium Bromide and can be examined under blue light/UV. We have used it in our laboratory.

Hi! I wanted to see if you ever got a working protocol for IL17a-Cre genotyping? I am having problems with the Jackson protocol as well.

Amy Klocko Thank you for the tip! Highly appreciate it.

Can it analyse eberhart and Russell model and how the significance of regression coefficient is calculated using GEA-R

Yes, overused TAE can contaminate the quality of your expected bands. It is recommended you keep your chamber clean all the time especially when you are working with different/new sets of samples.

You can also choose to study the diversity, evolutionary phylogenetics or domestication of a species. Large stractural varitions on genetic disease is also a choice.

The following is the illustration on input file format for multiple alleles for microsatellite(SSR) loci in diploids :

SSR1A SSR1B SSR1C SSR2A SSR2B........... so on

Genotype-1 1 0 0 0 1

Genotype-2 1 0 1 1 1

.

.

.

So on

hi Singh

I hope that enough information to answer

NCBI provides us information of a particular loci in any one strand (i.e., either forward or reverse). To elaborate, for example you have a variant change from T/C based on your sequence retrieved from NCBI/Ensembl (the sequence is given in reverse strand), but when you search for the variant in NCBI it shows A/G (forward strand). Both are correct highlighting the complimentarity. Minor and major allele remains the same. Yes. As previously mentioned you can refer to it as reference allele/mutant allele.

Hi Ahmad,

just to check I am reading the results correctly your A260/230 values are in blue and are around 0.3-0.4. If this is the case then something is absorbing at 230 . If you are using standard dna prep kits they use a lot of thiocyanate to act as a denaturant. If you use too much crude lysate or not enough wash solution this thiocyanate can wash off with the dna and if the 260/230 is too low ( more than 2.0 is good) then this may denature the taq enzyme in the pcr leading to zero /poor amplification. If you are using a commercial kit try loading less tail tissue or doing an extra wash step before eluting your dna

This is a good source of primer/probe for detection of FAdVs by rRT-PCR:

Brian Thomas Foley I was afraid of that answer but it seems indeed that's the only option.

Thanks for helping :)

Hi Gabriel,

one of mice that you are interested in is available from the Jackson Laboratory and they typically provide the genotyping information on their website under the technical support section of the specific mice.

It looks like you will have to perform two separate PCRs. The PCR information for C57BL/6-Il17atm1Bcgen can be found here (see technical support section):

For your other mouse strain (IL22promTdtomato BAC) you can easily check the original paper:

Generally it is good to check if your mice are available at the Jackson Laboratory. They typically provide the needed PCR information. If they can't be found there it is always best to look up the original paper that established the mouse model.

Patrick

For minimum spanning tree, you can use EdeNetworks.

yes, you can do it. you can choose with Genstat software too.

The reason is that "+" strand (sense) is considered as the default DNA strand in the most cases, whereas targets for probes for SNP array can be located on any strand, according to convenience for array design. So, you need to take into account strand information in order to avoid mistakes in interpretation of data.

Also, it seems like you confuse complementary base pairs with a couple of allelic variants. In your example ([A/T] and [T/A]) both bases are allelic variants on the same strand (check if the workflow included strand conversion). Their order in the genotype record depends on what allele is considered to be the reference, and what is the alternative. This information is contained in the manifest file of corresponding microarray.