Research Method

(a) Hierarchical sequencing

(b) Shotgun sequencing g

A human chromosome is stained to reveal its bands

^ One 5 million bp band is ^ removed and isolated for analysis.

DNA l

| The DNA is cut into large 55,000-2 million bp fragments by a restriction enzyme. The fragments are cloned in bacteria artificial chromosome (BAC) vectors

| Sequence-tagged sites (STS) are identified on the fragments; common ones indicate overlaps.

Human DNA i is randomly broken into 500-800 bp fragments.

Each fragment is sequenced.

The BAC fragments are cut into small pieces and sequenced from STS to STS, 500 bp at a time.

DNA l

| The DNA is cut into large 55,000-2 million bp fragments by a restriction enzyme. The fragments are cloned in bacteria artificial chromosome (BAC) vectors

| Sequence-tagged sites (STS) are identified on the fragments; common ones indicate overlaps.

The BAC fragments are cut into small pieces and sequenced from STS to STS, 500 bp at a time.

Plasma Membrane Images

Human DNA i is randomly broken into 500-800 bp fragments.

Each fragment is sequenced.

A computer finds overlapping sequences shared by a fragment, and the fragments are aligned.

17.21 Two Approaches to Sequencing DNA (a) In the hierarchical approach to genome sequencing, genetic markers are mapped, and the DNA fragments are then aligned by matching overlapping marked sites whose sequence is known (sequence-tagged sites,STS). (b) In the shotgun approach, the DNA is fragmented and a computer is used to find the markers.

are the marker sequences, and the "mileage" is in base pairs. The simplest markers are the recognition sequences for restriction enzymes.

Some restriction enzymes recognize 8-12 base pairs in DNA, not just the usual 4-6 base pairs. A DNA molecule with several million base pairs will have relatively few of these larger sites, and thus the enzyme will generate a small number of relatively large fragments. These large fragments can be added to a vector called a bacterial artificial chromosome (BAC), which can carry about 250,000 base pairs of inserted DNA, and inserted into bacteria to create a gene library.

The volumes (fragments) in this library can be arranged in the proper order along the chromosome map by using the marker sequences. To arrange the DNA fragments on the map, libraries made with different restriction enzymes are compared. If two large fragments of DNA cut with different enzymes have the same marker, they must overlap. This method works, but is slow.

shotgun sequencing. Instead of finding markers, fragmenting the DNA, and then sequencing it, the "shotgun" approach cuts the DNA at random into small, sequencing-ready fragments and lets powerful computers determine markers that overlap (Figure 17.21b). The fragments can then be aligned.

The shotgun sequencing method, which has been used by private industry, is much faster than the hierarchical approach because there is no need to make a map. At first there was considerable skepticism about this method. There were concerns that without rigorous prior mapping of marker sites on the chromosomes, the computer might pick out repetitive sequences common to many DNA fragments and line the fragments up incorrectly. But the rapid rate of development of sophisticated computers and software has allowed the shotgun method to be refined to a point at which inaccurate alignment is not a major problem. The entire 180 million-base-pair fruit fly genome (see Chapter 14) was sequenced by the shotgun method in little over a year. This success proved that the shotgun method might work for the much larger human genome, and in fact, it did.

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Essentials of Human Physiology

Essentials of Human Physiology

This ebook provides an introductory explanation of the workings of the human body, with an effort to draw connections between the body systems and explain their interdependencies. A framework for the book is homeostasis and how the body maintains balance within each system. This is intended as a first introduction to physiology for a college-level course.

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