Info

500,000

black > 66% AT; green > 64% AT.

m

The third circle shows coverage of some clones used in sequencing the genome.

The third circle shows coverage of some clones used in sequencing the genome.

Sma I 1,000,000

The fourth circle shows ribosomal operons (green), tRNAs (black) and |-like prophage (blue).

The fifth circle shows positions of simple tandem repeats.

■ Amino acid biosynthesis

■ Biosynthesis of cofactors, prosthetic groups, carriers H Cell envelope Cellular processes Central intermediary metabolism Energy metabolism Fatty acid phospholipid metabolism Purines, pyrimides, nucleosides and nucleotides Regulatory functions Replication H Transport/binding proteins IB Translation Transcription Other categories IB Hypothetical ITJ Unknown

19.9 The bacterium Haemophilus influenzae was the first free-living organism to be sequenced. (From R.D. Fleischman et al., 1993, Science 269:469; Scan courtesy of TIGR.)

After the genetic and physical maps are available, chromosomes or large pieces of chromosomes are separated by pulsed-field gel electrophoresis (PFGE) or by flow cytometry. In pulsed field gel electrophoresis (which is similar to standard gel electrophoresis), large molecules of DNA or whole chromosomes are separated in a gel by periodically alternating the orientation of an electrical current. In flow cytometry, chromosomes are sorted optically by size (I Figure 19.10).

Each chromosome (or sometimes the entire genome) is then cut up by partial digestion with restriction enzymes (iFigure 19.11). Partial digestion means that the restriction enzymes are allowed to act only for a limited time so that not all restriction sites in every DNA molecule are cut. Thus partial digestion produces a set of large overlapping DNA fragments, which are then cloned by using cosmids, yeast artificial chromosomes (YACs), or bacterial artificial chromosomes (BACs).

Next, these large-insert clones are put together in their correct order on the chromosome (see Figure 19.11). This assembly can be done in several ways. One method relies on the presence of a high-density map of genetic markers. A complementary DNA probe is made for each genetic marker, and a library of the large-insert clones is screened with the probe, which will hybridize to any colony containing a clone with the marker. The library is then screened for neighboring markers. Because the clones are much larger than the markers used as probes, some clones will have more than one marker. For example, clone A might have markers M1 and M2, clone B markers M2, M3, and M4, and clone C markers M4 and M5. Such a result would indicate that these clones contain areas of overlap, as shown here.

Ml M2 M4 M5

Clone A Clone C

M2 M3 M4 Clone B I

Ml M2 M3 M4 M5

Contig | III

A set of two or more overlapping DNA fragments that form a contiguous stretch of DNA is called a contig. This approach was used in 1993 to create a contig of the human Y chromosome consisting of 196 overlapping YAC clones (see Figure 19.3).

Chromosomes,

Cell

Chromosomes,

Cell

Cells are broken open to release chromosomes.

Chromosomes in dilute medium

Cells are broken open to release chromosomes.

Chromosomes in dilute medium

.and are stained with fluorescent dye.

.and are stained with fluorescent dye.

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