DNA sequencing by Maxam Gilbert method

DNA sequencing by Maxam Gilbert method |
Topic index:
1) End labeling
2) restriction enzyme digestion
3) denaturation
4) chemical degradation
5) gel electrophoresis
6) autoradiography
7) sequence determination
8) limitation
Maxam-Gilbert sequencing is a method of DNA sequencing developed by Allan Maxam and Walter Gilbert in 1976-1977. This method is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides.
Procedure:
Maxam-Gilbert sequencing requires radioactive labeling at one 5′ end of the DNA fragment to be sequenced (typically by a kinase reaction using gamma-32P ATP) and purification of the DNA. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T). For example, the purines (A+G) are depurinated using formic acid, the guanines (and to some extent the adenines) are methylated by dimethyl sulfate, and the pyrimidines (C+T) are hydrolysed using hydrazine. The addition of salt (sodium chloride) to the hydrazine reaction inhibits the reaction of thymine for the C-only reaction. The modified DNAs may then be cleaved by hot piperidine; (CH2)5NH at the position of the modified base. The concentration of the modifying chemicals is controlled to introduce on average one modification per DNA molecule. Thus a series of labeled fragments is generated, from the radiolabeled end to the first "cut" site in each molecule.
The fragments in the four reactions are electrophoresed side by side in denaturing acrylamide gels for size separation. To visualize the fragments, the gel is exposed to X-ray film for autoradiography, yielding a series of dark bands each showing the location of identical radiolabeled DNA molecules. From presence and absence of certain fragments the sequence may be inferred.
1) Preparation of Your Sample
The DNA used in Maxam-Gilbert sequencing is first denatured into a single-stranded chain, and labeled on the 5′ end, usually with 32P.
3) Electrophoresis + Autoradiography
The next step cleaves the DNA. And this is where the Maxam-Gilbert sequencing gets really interesting. By taking advantage of piperidine and two chemicals that selectively attack purines and pyrimidines (dimethyl sulfate and hydrazine, respectively), the DNA is cleaved at specific points. To be more accurate, using different combinations of these chemicals, you can cleave a DNA sequence wherever there is a C, wherever there is a C or a T; wherever there is a G or wherever there is a G or an A. So, if you put your sample into these 4 different reaction tubes, you obtain different fragments, depending on the combination of chemicals!
3) Electrophoresis + Autoradiography
These reactions are then loaded on to a high percentage polyacrylamide gel, to differentiate fragment sizes. The fragments are visualized via the radioactive tag.
4) Reading the Sequence
To read the sequence, you begin with the smaller fragments at the bottom of the gel. “Calling” each base involves interpreting the band pattern relative to the four chemical reactions. For example, if a band in the DNA sequence appears in both the G-reaction and the G+A-reaction lanes, then that the nucleotide is a G. If a band in the DNA sequence appears only in the G+A-reaction lane, then it is an A. The same decision process works for the C-reaction and the C+T-reaction lanes. Sequences are confirmed by running replicate reactions on the same gel and comparing the autoradiographic patterns between replicates.

[@creative_olive]

This is the best video I have seen on this topic so far. So detailing and comprehensive.

[@Muslimah_undercover_]

i took so many yt lects and did not understand and Alhamdhulilah i found this one its so clear word by word and all reactions wow

[@Radhaisspace]

It's my seminar topic .. thank you so much for helping me to understand the concept .and many people didn't explain about the chemical reagents but did ya .it is very helpful.

[@shahbaloch4814]

Great sir the best method of teaching very helpful agr sab teacher ap k esab say padae tho kohe subject mushkil nahe oga sab asan say samj ahe gay

[@sanasadique4473]

Sir mujha itni achi samj University mai b nai ai jitni achi apky lecture sy ai ha thankyou so much jazakAllah sir ☺ please asy hi lecture daty rahiya ga ham students k lia please

[@innocentokpako6195]

Wow! This is simply amazing... My 3 days+ search for clarity has just ended. Well done 👏

[@TheSingtangpaScienceGuy]

To the point , clear, crisp, simple, easy to understand.
You might also love to know that there is one particular University in India (whose name will be kept confidential) where one of the Genetics professors is giving notes to her students straight out of ur videos, as it is, without any modifications whatsoever. ;D
U shud b proud bruh!

[@TheSingtangpaScienceGuy]

Although i have a question on the G tube and A+G tube.
G is the first nucleotide in the example sequence u have used. When a cut is made, it will be made before G, meaning the first cut will produce only the radiolabelled phosphate without any nucleotide. But u still considered this a fragment and counted it as one fragment. Why is that?

[@elegantmobileaccessories1951]

Thank you for making me understood this concept..

[@husnashariff983]

Beautifully expalined✌️

[@bibliophilicnoor7531]

excellent sir...... now my concept is clear... n the credit goes to u

[@meenabarot2037]

excellent presentation!

[@muhammadyahya9055]

what an explanation and points extracted ...thanks a lot sir ...really big thanks ..

[@SpillFreakMusic]

Thank you. Very helpful.

[@microbioscope18]

Spill Freak 😂😂😂

[@manishasolanki3822]

Thnk you vry mch sir... It is much more helpful to me than other vdeos.. 😊

[@riichobamin7612]

It's an awesome video and I don't want to nitpick but, and I am really sorry for this, its "labelling".
Keep up the good work !

[@rajeshkumarsattenapalli5085]

Thank you very much

[@kumarsaurabhsingh2389]

I subcribed to ur channel.... I like it... Now I am commenting...
Bro... That was truly awesome...
U r a great teacher.... Respect from bottom of heart...

[@rabiaparvaiz9641]

Sir ur lectr is awsom

[@stephenlentsa7479]

10:00 I would like to ask, do we amplify our strand that we will be able to get those number of strands in each tube? As for that to be possible that means we have to have the same DNA strand in each tube. Thank you😊

[@microbioscope18]

Stephen Lentsa .... We take multiple copies of that DNA strand in each tube .

[@peepdi]

@@microbioscope18 that is done by PCR?

[@--HiroshiChawla]

@@peepdi Yes

[@dmariya1521]

Gd explanation sir

[@bioatbench350R]

I have a doubt regarding the 12th nucleotide..if piperidine cuts in C Nd C+T tube it will cut only at 5' of C thn it will become 11 nucleotide ..how it can be the last one my concepts isn't clear there ..thn according to me it lacks one nucleotide

[@hadiagul2732]

Thanku so such an amazing video

[@shabazquddus7791]

Sir, if we don't know the sequence of the DNA then how can we cut with restriction enzyme . As to cut with RE we must know the sequence of a restriction site ?

[@pritiprasannasahoo7804]

Very good presentation ...

[@samruddhikarale8592]

Sir very informative vedio and easy to understand..plz make vedio on automated DNA sequencing

[@dibyendyusaha1771]

If the first base will cut. Then how the dna fragments are formed after it?

[@daymohin6834]

I think that's why 32-p i's added,it will produce radioactivity at that region As it carries radioactive phosphate

[@delnarosedavis7674]

Multiple copies of the ssdna are added.

[@mulengae5]

Is the same as KDEL sequence?

[@rosebdg903]

Thanks I have understood the technique and continued on like this

[@pratibhasharma7246]

Thanks sir 🙏

[@shayannaeem1118]

Thanku so much sirrr..... Lots of prayers...u nailed it

[@animalslover865]

This is amazing my brother . Thank you .

[@peepdi]

Why can't we break adenine in the second test tube as we did in the case of guanine?

[@Mxyksykdkdodksodosirameshsingh]

thkxx u do good work
God bless u MERE DOST

[@rakshithm1257]

dude... you are the man!

[@WaqasKhan-sl3yh]

Why one or two nucleotide neglected during electrophoresis

[@Muslimah_undercover_]

you said only one cut will be made per fragment but then you said 6 or 2 fragments after multiple cuts will be produced can someone clear my confusion please

[@parathsahu1548]

Very nice sir

[@stephenlentsa7479]

And 13:10 how can we determine the length of the DNA strand on the electrophoresis gel?

[@microbioscope18]

We run marker DNA in one lane to get the idea about the length of DNA fragments . For more details at first follow videos on agarose gel electrophoresis to clear the concept .

[@subhiksharajan8547]

Superb ,thanks for ur clear lecture

[@peepdi]

I don't understand in the step where we are doing dephosphorylation why is hydroxyl group attached to 5 prime end of both the DNA strands that is a bit confusing

[@pihugumber6929]

Outstanding boss👌

[@yarakawasmi7403]

يسعد دينك اخيراً فهمت

[@akhlaqkhan3038]

Thanks sir

[@TheMato1112]

Very good video it helped me a lot thank you wish u luck with scientific projects :)

[@microbioscope18]

Matej Forgáč ... Most welcome ☺☺

[@ProfSardarMNiaz]

What is the purpose of 3rd step? I am not getting.
Suppose after end labelling we denature the DNA we will get end labelled ssDNA..
Then why we are cutting that DNA?

[@microbioscope18]

Sardar Zain ... Yes , that is an alternative method , i forgot to mention it but .....in some cases isolation of labelled ssDNA is more difficult by this method . May be that is the reason for including the restriction enzyme digestion step .

[@stephenlentsa7479]

This is because denaturing a whole DNA double strand requires more energy, so as to avoid human errors, the DNA has to be prepared into shorter fragments for easier electrophoresing

[@PujaKumari-rk9rg]

excellent & very helpful

[@microbioscope18]

Puja Kumari thanks 😊😊😊

[@shahbaloch4814]

Send the link of Sanger method

[@taniabatool4451]

Thank u

[@the_encephalonaire]

Can I do msc in genetic engineering after my Bsc in microbiology. Which goverment University in india allows msc in genetic engineering after bsc Hons in microbiology?

[@Ijazalijin]

how the same nucleotide will give us different bends in the same tube?

[@tanushanaik2568]

Sir can u tell me that in each tube is there many copies of ssDNA is present or for each tube only one strand is present? Can u clear this plz

[@coachpsr1724]

Thankew sir good job..very helpful..keep doing

[@microbioscope18]

most welcome

[@JyotiSharma-fz6zg]

so nice trick of explain sr thank you so much

[@microbioscope18]

Jyoti Sharma .... Most welcome ☺☺

[@JyotiSharma-fz6zg]

thanks for sporting us

[@veermaniprasad]

nice explanation

[@cherrina_mamikero6900]

Thank you🙏

[@sidrasanam3290]

Thanku sir.. it was very helpful .. but sir i have a question... if dna cut at restriction site and divided into two.. then the smallest one is discarded as you mentioned in the lecture.. so we will not get the sequence for that discarded part? We will get dna sequence for the longer part only..?

[@microbioscope18]

Sidra Sanam yes , if the fragment is very small compare to other fragment then we can discard the small fragment and we will not get the sequence of that..... Only sequence larger fragment .

[@sidrasanam3290]

subrata das thanks alot sir for the reply

[@dr.jyotisaxena6966]

Can 1st nucleotide be detected? If not, what is the reason?

[@iqramalik4659]

Sir in each tube there is one strand and one primer...if once primer cut strand at one position so this fragment separated with primer.then there is no other primer to again attach to template so how next chain start to make 2nd fragmnt???

[@microbioscope18]

iqra malik ... No , there are multiple copies of the DNA present and there is no primer .... Chemical reagents are there to cut the DNA .

[@iqramalik4659]

Sir actually my questn is about sanger method...by mistake I coment here...so plz clear it regarding sanger method

[@iqramalik4659]

subrata das reply must..

[@microbioscope18]

iqra malik .... Ok ... In sanger Sequencing there are multiple template DNA and primer is present ... I show only one to explain it easily

[@iqramalik4659]

subrata das manyy manyyy thankssss

[@sarah241lotus6]

you are amazing you saved my life thanks new subscriber

[@microbioscope18]

Sarah 241Lotus ... Most welcome

[@manthandambhare7395]

🙏Best explaination but sir if there are 12 nucleotide and C is removed by removing agent then largest sequence will be 11 isnt it?

[@Malaka1802]

You said that during degredation every ssDNA will only be cut at a single Position. I am afraid this is wrong. In reality a ssDNa can be cut multiple times. But since only one radioactive phosphor is introduced, you can only see those strands that contain the 5'ending with the radioactive phosphor. So it does not matter if the other end is cut any more since it is "invisible" to us.

[@nothing-gl2et]

u gave the perfect answer of my doubt . thanks

[@peepdi]

I have one more doubt why are we adding radioactive phosphate in both the DNA strands.. when we are only going to put one strand in each tube

[@zuhrimislam2210]

To determine the direction of the strand i.e 5' to 3'...As we know that the phosphate group is only exposed at 5' & not 3' end...So it will help us to pick the exact strand needed for this procedure which is 5' to 3' strand

[@kundandutta710]

Sir why we Have To elute out the heavier band and work with only the lighter band after the denaturation of DNA double-strand??
Our desired sequence may be in the the lighter band as well isn't it?
Please reply sir

[@sohelmahmudsohail3452]

where do you get this process??? i mean what is references????

[@sukritinarzary5388]

You stumble a lot Sir but still your way of teaching is very appealing 👍👍.

[@rohini698]

Its little confusing when u say its removes the nucleotide. The chemicals doesn't remove the nucleotide but it cuts at after that nucleotide.

[@IamVishal5]

Very good Sir.You have the capability to understand the students . But sir what is most modern technique to sequence DNA?

[@microbioscope18]

Next generation Sequencing (NGS)

[@64squaresdotcom62]

@@microbioscope18 oxford nanopore is the most advanced or most used is illumina

[@SavitaKumari-rm4md]

Based sequenced method is alse called maxam gilbert sequencing.... It is right or not

[@peepdi]

Also mention your references from where you have taught this to us so that we can know about it

[@muhammadhanif6589]

Nice

[@dnabiology6659]

How we know that dna fragment have 12 nucleotide which we take for experiment or how we know no. Of nucleotide in dna fragment which we take for experiment sir

[@microbioscope18]

Each band ( in different position) represents one nucleotide ....so total band number denotes total nucleotide number .

[@dnabiology6659]

@@microbioscope18 thanku sir

[@ankitachowdhury5268]

Sir first mey toh Apne Likha hai ki double stranded DNA...usko fir denature kiya.....fir uska sequence kyu banaya....o to replication sey bhi ho jata

[@shabbirahmed1201]

Amazing

[@nothing-gl2et]

nice explanatn ...please improve audio

[@adibsarwari]

hello sir, i have one question
in this sequencing why we use restriction enzyme to cut dna strand if we want to single strand

[@akhlaqkhan3038]

Because this enzyme cut DNA at specific sit

[@subhamkarmakar3320]

Awesome

[@parathsahu1548]

Sir Gene mapping video load kijiye na pls

[@poojabollaje9619]

Thankuuu...its helpfullll

[@IrshadKhan-rk3id]

🤩🤩🤩🤩🤩

[@RAJABABU-hf5ou]

Icont usd plzzz

[@anweshapal1019]

7:09

[@paktv1842]

Jo samjany wala part ta vo tu sahi sy samjaya nhi

[@iqramalik4659]

Plzzz replyyy

[@ayishamoideen1268]

chemical degradation not degredation

[@microbioscope18]

ayisha moideen .... Yaa ... It's a mistake 😁😁.... Good observation 👍👍

[@jacky6207]

amazing bbro, you saved me