How does Sanger Sequencing Work? - Seq It Out #1
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- Опубликовано: 16 июн 2015
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Let’s go back to the basics and explore the technology platform that has been regarded as the gold standard for many years. Yea, you guessed it! I am talking about Sanger Sequencing by capillary electrophoresis. Many might ask, “why is it called Sanger Sequencing?”
Sanger Sequencing is named after the inventor of this ground breaking technology, Dr. Frederick Sanger, who developed this method over 40 years ago in the mid-70s.
So, what are the basics of Sanger Sequencing?
It all starts by having a short primer binding next to the region of interest. In the presence of the 4 nucleotides, the polymerase will extend the primer by adding on the complementary nucleotide from the template DNA strand. To find the exact composition of the DNA sequence, we need to bring this reaction to a defined stop that allows us to identify the base of the very end of this particular DNA fragment. Sanger did this by removing an oxygen atom from the ribonucleotide. Such a nucleotide is called a dideoxynucleotide. This is analogous to throwing a wrench into a gear. The polymerase enzyme can no longer add normal nucleotides onto this DNA chain. The extension has stopped and we now need to identify what it is. We identify the chain terminating nucleotide by a specific fluorescent dye, 4 specific colors to be exact. Sanger sequencing results in the formation of extension products of various lengths terminated with dideoxynucleotides at the 3′ end.
The extension products are then separated by Capillary Electrophoresis or CE. The molecules are injected by an electrical current into a long glass capillary filled with a gel polymer. During CE, an electrical field is applied so that the negatively charged DNA fragments move toward the positive electrode. The speed at which a DNA fragment migrates through the medium is inversely proportional to its molecular weight. This process can separate the extension products by size at a resolution of one base. A laser excites the dye labeled DNA fragments as they pass through a tiny window at the end of the capillary. The excited dye emits a light at a characteristic wavelength that is detected by a light sensor. Software can then interpret the detected signal and translate it into a base call. When the sequencing reaction is performed in the presence of all four terminated nucleotides, you eventually get a pool of DNA fragments that are measured and separated base by base. What you will get in the end is a data file showing the sequence of the DNA in a colorful electropherogram and a text file which you can use to answer the questions you may be asking.
And that in a nutshell is Sanger Sequencing. 
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well-explained, short, comprising all steps = really nice video Thanks!!! :)
We are happy you enjoyed the video.
IS THAT A GOLD MICROPIPPETE !?
she's the 1% of lab techs
idk but I would probably break it within 5 months from dropping it
I thought it was a brass Harry Potter wand but I immediately thought that it’s was some sort of micropipette as well
The clear writing and the awesome drawings made my day
My goodness it really helped
Thankyou so much
I hope my exams would be better now
Thank you so much for this video! I've been stuck on basics while reading articles. This time, one video and boom - the concept is clear! I feel ready-to-use Sanger Sequencing in my workplace :D
Thank you for watching! We're doing all we can to take advantage of these opportunities to offer insight and illumination to others in the scientific community. Be sure to let us know if there's anything else you want to see from us.
Thank you for this short and targeted approach to answer the the sanger for those who haven't been through it practically.
Our pleasure! Thank you for watching.
I don't think I could have done my honours project without this channel.
Thanks for the easy to understand explanation and graphics. Helped me understand a procedure that took me weirdly long to get the concept.
Happy we could help!
Oh this video Is JUST perfect, answered all my questions the day before the big test, Thanks Natalie!
Glad we could help! Hope you nail that test.
So well done!!
00:00 Brillinat opening!!! Oh hey I didn't see ya there!!!
Thanks for watching.
Well done. Superb
Where can you buy a gold pipette? That is so cool.
God bless you.
THIS VIDEO IS FIRE YAS YOY GET IT GURL THANK YOU SO MUCH BLESS UR HEART ANGEL
Love the effort but can you *show* us?
In other words, can this whole process be perceived under a microscope and then recorded for us to see?
Or can it really only be 'seen' by the laser?
If so can we at least see how the laser does this?
It would be really helpful in understanding what's going on.
Very well explained. Thanks
Thanks for watching!
What happens to the 3 prime overhang after the ddNTP is added. Do you digest it before running on the gel? And if not, wouldn't this affect the migration of the DNA through the gel?
The DNA reaction is denatured prior to electrophoresis and run as single-strands. The strand with the 3' overhang does not contain a flourescent label, therefore is invisible to the laser during migration.
nice explained , i am great full to you . thank you
Thank you for watching.
how ti identify wheather the result is 5 the end or 3rd
I understand the theory 100% and how it's supposed to work. However, I don't know how well this will work in real life since there's such a tiny size difference.
Plus, how do you know you don't have it backwards?
Does this mean it can be used de novo ?
this sequencing is insane if you think about it
thanks, i finally got it!
This video is 10/10
Not quite. My mentor picked up on the fact that she uses gloves for drawing her explanations but doesn't when using a pipette
Neat!
I have a sequence chromatogram , I need to analayse it but how do I do that ?
If you have a CE sequencing file (.ab1 file), you can use free software to view the results (base calls). Sequence Scanner is one free software. To compare a file(or many files or specimens) to a reference, you can either print it and compare the A,T,C,Gs to a reference file or use software (free Variant Analysis on Thermo Fisher Scientific cloud).
That glass tube sorts by size...but you expect to get DNA sequence from it? Wouldn't sorting by size obfuscate the sequence?
Thanks for watching @DarkCarbunkle. Received your questions and will get back to you as soon as we can. Thanks.
How expensive is the equipment involved in Sanger Sequencing if I wanted to sequence my own DNA? Is it feasible for those that are not in the research field, but want to run their own experiments in a home-made lab? How about second-hand equipment? Thank you.
This depends on the number of sequencing bases (genomic region) you wish to explore. Going to your local core laboratory at a university or a commercial service provider would allow you to look at one stretch of ~600-1000 base pairs for about ~$3-9. If you own an instrument the reagent cost is about the same or a bit less. Home made lab.... not really feasible for automated fluorescent sequencing. Most instruments range from ~$40k-$300k. You can go used, however you do get what you pay for.
@@evennemeyer1 yes.. and additionally, this assumes that you have already prepared your dna such that it can be sequenced.. you will likely need to amplify the region of your DNA you want to be sequenced, using PCR - then either do a PCR cleanup or agarose DNA gel followed by gel extraction. At this point you will be ready to submit your sample for sequencing (you will also need to design primers for the region of interest, or select an in-house primer that's available from the sequencing facility)
Good
If we dont know the sequence of DNA, then how do we add primer?
Thanks for the question, Kalpana. In order to perform sanger sequencing, you would need to know a part of the sequence in order to design the primer. If you do not know any part of the sequence, a better alternative would be to perform Next Gen Sequencing. If you have any additional questions, please feel free to email techsupport@thermofisher.com.
The DNA in the capillary should be drawn single stranded! Because only the new strands are labeled and the template strands might be huge or even not .bef homogeneously sized, doulble strand electrophoresis would give non-interpretable results!
good
I have a question. Here we have seen what are the dideoxynucleotides that are at the end of a DNA fragment? However, what about the identification of each nucleotides in a single DNA fragment?
Oh my goodness , same question !
when the laser excites the dye bound to a dideoxynucleotide, a light sensor tells the computer what wavelengths it saw and the computer processes it and reports it as a color, which then gets reported as whatever base you told it goes to that color. (I.e., you'd tell the computer that A's have red dye) and it will print the order of the nucleotides based on colors as the tube moves across the sensor.
@laurelcook9078 that didn't answer the question though. Is the only relevant nucleotide of each fragment the dideoxynucleotide then?
Hi! How can we use a primer if we don't know the dna sequence?
Thank you for your question. Please contact our technical support team at thermofisher.com/askaquestion for additional information. Thank you!
Hi C A, usually you are trying to sequence a known fragment of DNA and you know the "flanking" sequence around your target. Alternatively, if the target sequence has been repackaged in a "vector", the vector has built in sequencing primer locations (like M13)
nobody talking about the GOLD PIPETTE
I still don't get how does the software know which ddNTP is the start of the sequence.
I get it will be one of the shortest fragments. But how do we know the shortest fragment is the 1st when it's random for all 4 ddNTPs
She said that when the strands move though the gel their speed is inversely proportional to the molecular weight, the shortest strand will travel the fastest and will be detected first.
@@ecneicsPhD4554 Thanks 👍
What was she pipetting?
Why am i still not getting it
How can we know the primer sequence without knowing the sequence of the DNA???
You can use a restriction enzyme to digest the DNA and then ligate it into a cloning vector and use the priming site to sequence the DNA.
From that point, you can perform a search in a public database like NCBI or you should have enough information to perform primer walking and sequence the DNA.
For additional technical questions, please contact us at thermofisher.com/askaquestion.
I honestly couldn't understand were the DNA goes. Where do you put it????
When you isolate the DNA, you place it into either PCR strip tubes, 96-well, or 384-well plates. The DNA is mixed with water and our BigDye Terminator Ready Reaction mix which contains the dNTP and ddNTPs , buffer and enzymes needed for the reaction, placed into a thermal cycler to allow for the reaction shown in the video to take place. The DNA is cleaned up to remove any of the ddNTP that have not been incorporated into the product and then placed on the capillary electrophoresis instrument, where it is injected, run and the data collected. If this is not the information you are looking for can you please clarify at which step you are asking about - is it before the reaction, what do you do with it after the reaction, where do you put it on the instrument, or where does the DNA go after moving in front of the laser?
she has a gold Pipette?
How did he remove that oxygen atom from just "one" infinitesimally-small molecule?
Thank you for your questions.
There are deoxynucleotides (dNTPs) and dideoxynucleotides (ddNTPs). The ddNTPs lack a hydroxyl group (so there is -H instead of -OH at a specific location in the structure of the nucleotide). (The creation of a ddNTP is performed on multiple ddNTPs, not one at a time.) Without that oxygen in the hydroxyl group, a phosphodiester bond can no longer be made by DNA polymerase if the polymerase should happen to encounter another nucleotide.
A sequencing reaction contains both dNTPs and fluorescently labeled ddNTPs. Using a DNA strand as a template, a complementary primer anneals to the template and DNA polymerase extends the complement in the 5’ to 3’ direction as complementary dNTPs are encountered. If a complementary dye-labeled ddNTP is encountered, the extension by DNA polymerase stops and a strand of a certain length is created with a dye label at one end. So
during each cycle, multiple extension products are created. These extension products will be various lengths because the dye-labeled ddNTPs will be incorporated at different times among the different templates. At the end of the cycles, there will be multiple fluorescently-labeled fragments.
Those fragments are then electrophoresed through a capillary. The smaller the fragment, the faster it will travel through the capillary. For example, a fragment that is 1oo nucleotides in length will travel faster than a fragment that is 101 bp in length. There will ultimately be fragments (all one base pair longer than the previous fragment that travel past a laser. The laser causes the fluorescent dye labels to fluoresce. Each ddNTP (A,C,G,or T) has a specific dye color associated with it and as the dye fluoresces, the color that emits is captured by a camera and base calls are made in the software based on the order of the colors.
If you have any additional questions as to how this works, please contact abtechnicalsupport@thermofisher.com. Thank you.
science babes yeahhh
The discover of a new genre Science Milfs.. hahah :)
gd llooks, from 8 years into the future
So you do it with lasers huh?
im related
She's not talking all that fast.
The DNA fragments going through the column should be single stranded, not double stranded.
You fucking legend, I could honestly marry you right now.
so........ she's kinda super pretty
How are we designing a primer when we don't even know the sequence?
hank you for your question Santam.
You can use a restriction enzyme to digest the DNA and then ligate it into a cloning vector and use the priming site to sequence the DNA.
From that point, you can perform a search in a public database like NCBI or you should have enough information to perform primer walking and sequence the DNA.
For additional technical questions, please contact us at thermofisher.com/askaquestion.
man you talk fast
Is she still married? You know, it doesn't matter. My real question is, can I give her my number? 😉