DNA Sequencing Using BAC and Shotgun Methods

How Scientists Sequence a Genome to Find the Genetic Code

Nov 9, 2009

DNA Sequencing Methods Determine Base Pair Order, mujitra (´???)

Genome sequencing is a way to determine the exact order of all the nucleotide bases, A, T, G and C, in a string of DNA using the BAC and Shotgun Sequencing Methods.

Scientists use DNA sequencing for a variety of projects, ranging from basic research on disease to forensic and paternity applications. The steps involved in sequencing a genome are complicated, but the process has become an invaluable tool in modern biological research.

What is DNA Sequencing?

All DNA is made up of a string of nucleotide bases, the chemical building blocks often referred to as the letters A, T, G and C. DNA sequencing is the process for figuring out the exact order of the nucleotide bases in a genetic code. In a human genome, there are over 3 billion nucleotide bases, so DNA sequencing can be a long and complicated process.

How Do Scientists Sequence DNA?

In the basic process of DNA sequencing, there are three major steps. First, the genome must be first broken up into smaller pieces, a process called subcloning. These small pieces range from 50 million to 250 million bases long.

Next, each short piece is used to make fragments that are slightly different lengths, differing by only one base. The fragments are run on an electrophoresis gel and separated.

Finally, the fragments are lined up and put back together, revealing the entire genomic sequence. Overlapping sections of each piece and matching up the areas that are the same lets the scientists determine the correct order. Modern labs have automated sequencers that can carry out this formerly labor-intensive step via computer.

BAC-to-BAC DNA Sequencing and Genome Shotgun Sequencing Methods

There are two specific methods that scientists perform the actual sequencing of a genome, BAC-to-BAC and shotgun sequencing.

The BAC-to-BAC method, also called the map-based method, is a well-established method of sequencing genes, but it tends to be quite slow. This method uses pieces of approximately 150,000 bases in the subcloning step, growing many copies of each piece in a circular structure called a BAC that is inserted into fast-growing bacteria and then pulled back out. Then, these 150,000 base pieces are further broken into pieces about 500 and 1500 bases long for sequencing.

For BAC-to-BAC, the scientists must perform a mapping step, where they map the chromosomes and the location of DNA on those chromosomes, before doing the actual sequencing of the DNA. After the DNA sequencing is complete, they rearrange all of the fragments back in the right order using the map as a guide.

The genome shotgun sequencing method is much faster and uses much smaller pieces of about 2000 to 10,000 bases. Scientists using the shotgun method can sequence an entire genome at once by breaking it into tiny pieces and then reassemble the pieces into the entire genome when finished without a map. The pieces are assembled like a jigsaw puzzle, using overlapping sections of the DNA itself instead of using a pre-determined map.

In both methods, computers are usually used to fit the final pieces back into a coherent structure. The Human Genome Project, which sequenced all of the bases in human DNA, used both methods and combined the data to generate the final human genome sequence.

Why is DNA Sequencing Important?

DNA sequencing can only give the specific order of bases in DNA. It cannot tell scientists what any part of the genetic code actually does. However, sequencing can be useful in many areas of biology and gives scientists a template for determining how genes and cells work in the body.

A few of the major uses of DNA sequencing are:

Diagnosing Diseases – Comparing normal sequences to sequences in people with genetic illnesses to determine what parts of the genome are involved in the disease.

Forensic Genetics – Comparing DNA left at a crime scene to the DNA sequence of suspects or victims

Paternity Tests – Matching the DNA of parents and children to determine how they are related.

Comparisons With Other Genomes - Comparing genomes of different species to help in scientific research and conservation efforts.

The technique of DNA sequencing holds great promise in generating information that will guide scientists to learn more about how the genome affects health, evolution and the genetic relationships between individuals and species.

Sources:

Venter, J.C. et al. A new strategy for genome sequencing. Nature 381, 364-366 (May 30, 1996).

The National Human Genome Research Institute. U.S. Government Website.

The copyright of the article DNA Sequencing Using BAC and Shotgun Methods in Genetics & Evolution is owned by Bridget Coila. Permission to republish DNA Sequencing Using BAC and Shotgun Methods in print or online must be granted by the author in writing.