Dna is a long polymeric molecule. Much like a ladder. Thats been twisted. The rails of this ladder are made up of sugar and phosphate groups under rungs, a pair of compounds known as nucleotide bases or base pairs, as we refer to them. Do you recall the name of the four nucleotides that make up dna? They are adenine, cytosine, guanine and thymine. These bases hydrogen bond together in defined patterns a to t and c to g in a cell. It is the specific order or sequence of these base pairs along this sugar phosphate backbone. That dictates what amino acids and proteins that cell can create. You may recall from lectures that a change in a single base pair in a cell can cause a malfunctioning protein and thereby cause disease in this video im going to talk you through some of the technologies that are now being used to study dna sequence at an Unprecedented scale, when we study a sample here at the broad, it is generally to ascertain the sequence of these base pairs in a sample of dna. Hence we call it dna sequencing. The technologies used for dna sequencing have undergone an amazing transformation in the last decade, or so. I will now walk you through some of the technologies that have been used historically and some of the newer ones that we are using today. So when did the modern age of dna sequencing begin well, our story starts back in the 1970s when scientists developed methods to label fragments of dna using radioactivity, fred sanger, a scientist working in the uk developed a method that ultimately proved to be the most scalable and Automation friendly method: this method still bears his name today.

So how did this signer method work? Well, in sagar method, we start with a strand of dna. Thats been split along the length, so its single stranded. Imagine cutting that ladder down the middle in the presence of a dna copying enzyme or dna polymerase and a primer molecule. That is a piece of dna. Complementary to one section of your single stranded dna dna polymerase will add free nucleotides in the solution to your growing strand of dna. Nucleotides are added to the three prime end of that primer molecule according to base pairing rules. So an a will be added to a t on the template. A g would be added to a c on the template in the sanger method. A small number of these free floating nucleotides in solution are chemically modified so that, when theyre incorporated by the dna polymerase molecule, no further incorporation events can happen. These modified nucleotides are called dideoxynucleotides. Dna polymerase will continue to incorporate bases from solution until it randomly incorporates. One of these modified bases, which terminates the reaction by repeating this cycle over and over many times, we end up with a pool of fragments that each differ in length by a single base until every base of your original template molecule is represented in the early days Of the sagger method, scientists would set up four individual reactions in each reaction. One of the four bases, a t c or g – was present in both modified and unmodified forms.

The other three bases were normal. Products from these reactions were then loaded onto a gel matrix and a charge was applied because dna contains a charge it would pass through the gel matrix. Smaller molecules will pass through that matrix faster than larger molecules. If the gel is sufficiently long, you get single base resolution such that those templates that start at the beginning of your dna sequence will migrate the fastest and hence go to the bottom of the gel. Whereas the longest molecules which represent the entire length of the sequence, would travel the slowest and end at the top of the gel because they use radioactive labels, scientists could then expose this gel to an x ray film, which would record the position of each of the Fragments in that gel, the radio label method was quickly replaced by fluorescent dyes, as these were far easier to use and instead of doing four reactions, all four bases could be interrogated in a single reaction by using four different dyes. The first instrument to automate this process was commercialized by a company called applied biosystems in 1987.. In this technology they replaced the glass plates used to pour gels with a series of thin glass capillaries, and this became known as capillary sequencing. This technology can process up to 96 individual sequencing reactions at once. The human genome project, which started in 1990 largely used this technology. The highest throughput version of the capillary sequencing technology from applied biosystems was the 3730 model that you see here.

This technology, which uses sanger sequencing, could process up to 96 individual sequencing reactions at once at a cost of a couple of dollars per reaction. These thin glass tubes contain a gel matrix through which the dna fragments travel and resolve by size before passing through a laser beam at the end which interrogates, which fluorescent dye is present on the fragment of dna and thereby which base is present at that location. In the dna, so what came next in sequencing technology? Well, in the early 2000s, several groups were working on new molecular and technological methods to drastically increase the number of sequencing reactions that could be performed at once. The first such technology to come to market came from a company called 454 life sciences in 2004 in 454. Sequencing dna is first fragmented into small chunks about three to four hundred bases. To these fragments, small sections of known dna sequence are added to each end. These sequences are called adapters. You can think of these like the little forks that you stick into the end of a corn cob, so that it can more easily be handled, manipulated and moved, and molecular adapters perform much the same function adapted dna fragments are then added to the surface of tiny Beads we dilute the fragments to promote a ratio of one b to one piece of dna on the surface of these beads are binding sites for one of the adapter ends of your dna fragment.

So, at the end of this process, you end up with about a million copies of that dna template on the surface of each bead in the 454 pyrosequencing method, microbeads with amplified dna on the surface are first placed into tiny wells on the surface of a glass Slide known as a pico tighter plate when a nucleotide is flowing across the surface of this plate and incorporates to a single stranded piece of dna on the surface of a bead, a chemical reaction is kicked off an enzyme cascade that results in the release of a Flash of light, a very sensitive camera pointed at the peak or tider plate can record where, on that plate, the flash of light has occurred and thereby infer what base has just been incorporated into the growing strand of dna. In contrast to capillary sequencers, the 454 instrument could initially process tens of thousands and ultimately, in the model, you see up to a million sequencing reactions at once, all contained within the tiny pores. On the surface of this picotighter plate thats a 10 000 fold increase in scale which ushered in a new era in dna sequencing. So what is the highest? Yielding dna sequencing instrument in operation at the broad today, that is the high seek instrument from a company called illumina. The illumina technology starts with a dna fragment prepared in essentially the same way as a 454 sample, instead of using tiny beads to isolate and amplify the dna.

This sample is instead flown across the surface of a glass slide that contains a lawn of sticky dna fragments. These fragments bind to the adapters on the end of the dna sample bound fragments are then amplified in the spot that they bind on the surface of the glass slide to create a little cluster of fragments all with the same sequence, fluorescently labeled bases are added in A reaction that is very similar to the original sanger method. Once a tag base is incorporated, a laser excites, the fluorescent tag and a camera takes a picture of the surface of the glass slide. Then the terminating group of the base is removed and another set of tagged bases are added, another picture is taken and so on and so on until the entire fragment is red. This technology today can record up to 6 billion thats billion, with a b sequencing reactions at once, thats over 60 million times more than the capillary sequencer. This technology is capable of producing dna sequence, data equivalent to one human genome every day, easily at a cost thats about 100 000 times cheaper. When you consider that the first human genome took around 15 years and three billion dollars to complete, you can see. Why were all so excited about these technologies? So has technology development in the sequencing world stopped or slowed down? Absolutely not. There are several new sequencing technologies in various stages of maturity. The one that you can see here is the personal genome machine from a company called ion torrent.

This technology uses microprocessors similar to those found in your computer to detect the incorporation of dna bases, because it does not need a laser or camera. This instrument can be much smaller, faster and cheaper than the high throughput instruments. This personal genome machine can process five to seven million sequencing reactions at once in just about two hours. This other instrument, a single molecule sequencer from pacific biosciences, is quite different. Unlike the other sequencing technologies, it does not require one to make copies of the dna fragment before sequencing, providing technical advantages for some applications. This technology utilizes finely engineered stages on top of which a single dna polymerase molecule is attached the machine records in real time the incorporation of fluorescently tagged bases by this fixed dna polymerase. The length of the dna sequence. Output by this instrument is longer than that of other technologies up to several thousand bases. The number of parallel reactions is lower, however, only in the hundreds of thousands okay, so i really hope you enjoyed our tour today through the recent and not so recent history of the technologies used to sequence. Dna.

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