DNA sequencing?

Question

so what is the purpose of DNA sequencing, and how is it done?

My basic understanding is that we can determine the order of the 4 bases in a fragment of DNA somehow using dideoxynucleotides, which stop synthesis because of a missing hydroxl group. I know that the human genome project has something to do with this process, but im confused what the connection is there.

explain in detail if you can, but most importantly i want to know the purpose and basic concept of DNA sequencing and the HGP.

jamaica you rule! i would still like to know, what this has to do with HGP!

Answer 1

DNA sequencing reactions are just like the PCR reactions for replicating DNA (refer to the previous page DNA Denaturation, Annealing and Replication). The reaction mix includes the template DNA, free nucleotides, an enzyme (usually a variant of Taq polymerase) and a 'primer' - a small piece of single-stranded DNA about 20-30 nt long that can hybridize to one strand of the template DNA.
The reaction is initiated by heating until the two strands of DNA separate, then the primer sticks to its intended location and DNA polymerase starts elongating the primer. If allowed to go to completion, a new strand of DNA would be the result. If we start with a billion identical pieces of template DNA, we'll get a billion new copies of one of its strands.

Dideoxynucleotides: We run the reactions, however, in the presence of a dideoxyribonucleotide. This is just like regular DNA, except it has no 3' hydroxyl group - once it's added to the end of a DNA strand, there's no way to continue elongating it.
Now the key to this is that MOST of the nucleotides are regular ones, and just a fraction of them are dideoxy nucleotides....

Replicating a DNA strand in the presence of dideoxy-T
MOST of the time when a 'T' is required to make the new strand, the enzyme will get a good one and there's no problem. MOST of the time after adding a T, the enzyme will go ahead and add more nucleotides. However, 5% of the time, the enzyme will get a dideoxy-T, and that strand can never again be elongated. It eventually breaks away from the enzyme, a dead end product.

Sooner or later ALL of the copies will get terminated by a T, but each time the enzyme makes a new strand, the place it gets stopped will be random. In millions of starts, there will be strands stopping at every possible T along the way.

ALL of the strands we make started at one exact position. ALL of them end with a T. There are billions of them ... many millions at each possible T position. To find out where all the T's are in our newly synthesized strand, all we have to do is find out the sizes of all the terminated products!

Here's how we find out those fragment sizes.
Gel electrophoresis can be used to separate the fragments by size and measure them. In the cartoon at left, we depict the results of a sequencing reaction run in the presence of dideoxy-Cytidine (ddC).

First, let's add one fact: the dideoxy nucleotides in my lab have been chemically modified to fluoresce under UV light. The dideoxy-C, for example, glows blue. Now put the reaction products onto an 'electrophoresis gel' (you may need to refer to 'Gel Electrophoresis' in the Molecular Biology Glossary), and you'll see something like depicted at left. Smallest fragments are at the bottom, largest at the top. The positions and spacing shows the relative sizes. At the bottom is the smallest fragment that's been terminated by ddC; that's probably the C closest to the end of the primer (which is omitted from the sequence shown). Simply by scanning up the gel, we can see that we skip two, and then there's two more C's in a row. Skip another, and there's yet another C. And so on, all the way up. We can see where all the C's are.

Putting all four deoxynucleotides into the picture:
Well, OK, it's not so easy reading just C's, as you perhaps saw in the last figure. The spacing between the bands isn't all that easy to figure out. Imagine, though, that we ran the reaction with *all four* of the dideoxy nucleotides (A, G, C and T) present, and with *different* fluorescent colors on each. NOW look at the gel we'd get (at left). The sequence of the DNA is rather obvious if you know the color codes ... just read the colors from bottom to top: TGCGTCCA-(etc).

(Forgive me for using black - it shows up better than yellow).

An Automated sequencing gel:
That's exactly what we do to sequence DNA, then - we run DNA replication reactions in a test tube, but in the presence of trace amounts of all four of the dideoxy terminator nucleotides. A gel separates the resulting fragments by size and we can 'read' the sequence from it, bottom to top.

In a large-scale sequencing lab, we use a machine to run the gels and to monitor the different colors as they come off the bottom. It's called (not surprisingly) an automated DNA sequencer. There's an ultraviolet laser built into the machine that shoots through the gel near the botton and scans side to side, checking for bands of fluoresceent colors to pass through its beam. There might be as many as 96 'lanes' of samples running in one gel. Here's what a real fragment of a gel looks like (at left). The four colors red, green, blue and yellow each represent one of the four nucleotides.

The actual gel image, if you could get a monitor large enough to see it all at this magnification, would be perhaps 3 or 4 meters long and 30 or 40 cm wide.

A 'Scan' of one gel lane:
We don't even have to 'read' the sequence from the gel - the computer does that for us! Below is an example of what the sequencer's computer shows us for one sample. This is a plot of the colors detected in one 'lane' of a gel (one sample), scanned from smallest fragments to largest. The computer even interprets the colors by printing the nucleotide sequence across the top of the plot. This is just a fragment of the entire file, which would span around 700 or so nucleotides of accurate sequence.

The sequencer also gives the operator a text file containing just the nucleotide sequence, without the color traces.

DNA sequencing, the process of determining the exact order of the 3 billion chemical building blocks (called bases and abbreviated A, T, C, and G) that make up the DNA of the 24 different human chromosomes, was the greatest technical challenge in the Human Genome Project. Achieving this goal has helped reveal the estimated 20,000-25,000 human genes within our DNA as well as the regions controlling them. The resulting DNA sequence maps are being used by 21st Century scientists to explore human biology and other complex phenomena.

There are some experimental techniques being developed now that are quite different (resequencing based on hybridization to some kind of probe being one I've heard of), but today, if you do sequencing, you are using some kind of capillary sequencer, probably from ABI, and it works as I described above.



The human genome reference sequences do not represent any one person’s genome. Rather, they serve as a starting point for broad comparisons across humanity. The knowledge obtained is applicable to everyone because all humans share the same basic set of genes and genomic regulatory regions that control the development and maintenance of their biological structures and processes.


What is swbarnes2 talking about? Looser!!!

Answer 2

As for sequencing the other two responses are good. For the HGP the goal was to "read" the whole order of the DNA from a human, about 3 billion letters.

Once we have the DNA "read" we need to understand what it means that is the harder part. Within the 3 billion letters of DNA are the codes to make about 25,000-35,000 proteins. Each of those proteins serve a function in our bodies.

Many human diseases are caused when one or more of those protiens is not working correctly or is missing. By reading the DNA we can use computers to identify the codes for those proteins and understand specifically what is broken or missing in a person with a genetic disease. Eventualy when we understand what is broken, we can attempt to "fix" it with drugs or other treatments. Without the information of the DNA it is difficult to know what exactly is "missing" or broken, and finding a cure would be much more difficult.

Reading the DNA itself was just one step of the process, understanding it, and then using that knowledge to solve human problems is much slower, we are just beginning to see the earliest benefits of knowing the human DNA from the HGP.

Answer 3

DNA sequencing is basically determining the sequence of the nucleotide bases yet DNA replication comprises the possibility of transcription and translation (with the intention to accomplish replication you ought to do sequencing) the way PCR works is which you're taking the DNA and heat it upto ninety 5 levels to interrupt down the hydrogen bond then save a replica of the unmarried strands then you relatively take the newly replicated dna warmth it up , reproduction it and do the technique as quickly as greater

Answer 4

DNA sequencing is the determination of the precise sequence of nucleotides in a sample of DNA.

The most popular method for doing this is called the dideoxy method or Sanger method (named after its inventor, Frederick Sanger, who was awarded the 1980 Nobel prize in chemistry [his second] for this achievment).

The DNA to be sequenced is prepared as a single strand.

This template DNA is supplied with
a mixture of all four normal (deoxy) nucleotides in ample quantities
dATP
dGTP
dCTP
dTTP

a mixture of all four dideoxynucleotides, each present in limiting quantities and each labeled with a "tag" that fluoresces a different color:
ddATP
ddGTP
ddCTP
ddTTP
DNA polymerase I

Because all four normal nucleotides are present, chain elongation proceeds normally until, by chance, DNA polymerase inserts a dideoxy nucleotide (shown as colored letters) instead of the normal deoxynucleotide (shown as vertical lines). If the ratio of normal nucleotide to the dideoxy versions is high enough, some DNA strands will succeed in adding several hundred nucleotides before insertion of the dideoxy version halts the process.

At the end of the incubation period, the fragments are separated by length from longest to shortest. The resolution is so good that a difference of one nucleotide is enough to separate that strand from the next shorter and next longer strand. Each of the four dideoxynucleotides fluoresces a different color when illuminated by a laser beam and an automatic scanner provides a printout of the sequence.