Looking Closer: DNA Sequencing
A Closer Look...
On the Background page, the genome sequencing process is briefly discussed but is not explained in complete detail. Here you can you learn more about the real process behind how scientists are able to sequence the genomes of organisms and microorganisms alike.
DNA Sequencing Process
Step 1: Preparing the DNA
The very first step in DNA sequencing is to replicate the unknown sequence multiple times to get billions of copies. To do so, a section of DNA has its strands separated (through denaturation) and each strand gets a primer. DNA polymerase is added and it creates a exact complement (the "other half" of the DNA strand) as it travels down the strand. In one double-strand of DNA, this process will create two identical pieces. This is repeated until there are billions of copies to work with. L.1, l.1
Here on the left is an diagram of DNA replication.The DNA is split into separate strands and then complementary strands are built using the original strand. Notice how the resulting DNA is a combination of both the newly built strand and the original strand.
Step 2: Measuring the DNA
DNA replication is cloning the information, but it is not like a copy machine making tons of copies of the same thing. For instance, some of the nucleotides are dideoxynucleotides. Both the dideoxynucleotides and the addition of Thymine bases will terminate the strand. As a result of both the dideoxynucleotides and the Thymine bases, there will be different sized fragments of DNA. To find out where all the Thymine bases are within the sequence, running a gel electrophoresis will separate the DNA fragments according to size. L.1
Here on the left is an diagram of gel electrophoresis. Smaller fragments travel farther away from the wells than larger ones. In order to be able to identify the different bases, they are dyed with
fluorescent chemicals.
By knowing the size and the base, the sequence can be written out. This process is also done by machine with large pieces of DNA fragments.
Genome Sequencing: Dealing with Gigantic Pieces of DNA (L.1)
The process of DNA sequencing described above will only work with about 900 nucleotides. For organisms such as humans with over a billion bases, the genome has to be broken up into smaller pieces for the sequencing process to work. This can be done using BACs (this process was actually used to sequence the human genome).
By creating a "library" of BACs, each BAC can carry a large piece of the genome that is easier to sequence. There are often multiple copies of the same gene within different BACs due to random overlap when the BACs were originally cut. This creates an almost infinite supply of DNA fragments to sequence, allowing room for error and allowing the genome to be combined by identifying overlapping bases. An example of how a BAC library is created is shown on the left.
To the left is a diagram of the shotgun sequencing process. In shotgun sequencing, millions of copies of BACs are cut up randomly and the resulting fragments are placed into plasmids. The bacteria with the BAC-carrying plasmids are then cultured and the insert is sequenced. This is done until the original BAC is sequenced based on overlapping fragments.
Basic Definitions to Know
Deoxyribonucleic Acid (DNA): Contains all genetic information needed for an organism to survive. It is in the form of a double helix (twisted ladder), and can be split apart into separate strands and analyzed from there.
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DNA Polymerase: A specific enzyme (type of protein) that analyzes the bases on each strand of DNA and creates a complementary strand from the original.
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Denaturation: The process of changing the shape (and possibly makeup) of a protein, generally by heating.
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Primer: A short piece of DNA that sticks to one end of the strand.
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Dideoxynucleotides: They are similar to the DNA nucleotide, but do not contain a 3' hydroxyl group, and therefore cannot be elongated (strand ends there).
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Thymine: One of the DNA nucleotide bases. It pairs with Cytosine, and generally ends a strand of DNA.
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Gel Electrophoresis: A process of separating DNA. Samples are loaded in to the wells of a gel, and the whole gel is placed in a solution that allows for electricity to flow through the gel. Because DNA is negatively-charged, it will travel with the current across the gel, thus separating the DNA fragments. L.2
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Bacterial Artificial Chromosome (BAC): Similar to a plasmid, but is created by scientists to give a host bacterium a certain piece of DNA. This becomes very useful for genome sequencing, due to a bacteria's ability to clone itself.
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Shotgun sequencing: A method to determine the sequence of a large piece of DNA, by fragmenting the DNA and inserting the smaller pieces into a vector for replication. The results are analyzed to find out what the original BAC DNA sequence was.
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Plasmid: Small, circular loop of bacterial DNA that is generally used to carry and exchange genetic material between bacterial via binary fission (since bacteria reproduce asexually). L.3
Insert: Piece of DNA that has been inserted into another piece/section of DNA. It is an unknown sequence, and inserting it into a known sequence allows for analysis or determining what the sequence is.