1. Fig 18-1 Figure: 18-01 Caption:
The restriction enzyme EcoRI recognizes and binds to the palindromic nucleotide sequence GAATTC. Cleavage of the DNA at this site produces complementary single-stranded tails. These single-stranded tails anneal with single-stranded tails from other DNA fragments to form recombinant DNA molecules.

2. Fig 18-2 Figure: 18-02 Caption:
DNA from different sources is cleaved with EcoRI and mixed to allow annealing to form recombinant molecules. The enzyme DNA ligase then chemically bonds these annealed fragments into an intact recombinant DNA molecule.

3. Fig 18-3 Figure: 18-03_a Caption:
Some common restriction enzymes, with their recognition sequences, cutting sites, cleavage patterns, and sources.

4. Fig 18-3 Figure: 18-03_b Caption:
Some common restriction enzymes, with their recognition sequences, cutting sites, cleavage patterns, and sources.

5. Fig 18-3 Figure: 18-03_c Caption:
Some common restriction enzymes, with their recognition sequences, cutting sites, cleavage patterns, and sources.

6. Fig 18-5 Figure: 18-05 Caption:
The plasmid pUC18 offers several advantages as a vector for cloning. Because of its small size, it accepts relatively large DNA fragments for cloning; it replicates to a high copy number, and has a large number of restriction sites in the polylinker, located within a lacZ gene. Bacteria carrying pUC18 produce blue colonies when grown on media containing Xgal. DNA inserted into the polylinker site disrupts the lacZ gene; this results in white colonies and allows direct identification of colonies carrying cloned DNA inserts.

7. Fig 18-8 Figure: 18-08 Caption:
Phage lambda as a vector. DNA is extracted from a preparation of lambda phage, and the central gene cluster is removed by treatment with a restriction enzyme. The DNA to be cloned is cut with the same enzyme and ligated into the arms of the lambda chromosome. The recombinant chromosome is then packaged into phage proteins to form a recombinant virus. This virus infects bacterial cells and replicates its chromosome, including the DNA insert.

8. Fig 18-10 Figure: 18-10 Caption:
The cosmid pJB8 contains a bacterial origin of replication (ori), a single cos site (cos), an ampicillin resistance gene (amp, for selection of colonies that have taken up the cosmid), and a region containing four restriction sites for cloning (BamHI, EcoRI, ClaI, and HindIII). Because the vector is small (5.4 kb long), it can accept foreign DNA segments between 33 and 46 kb in length. The cos site allows cosmids carrying large inserts to be packaged into lambda viral coat proteins as though they were viral chromosomes. The viral coats carrying the cosmid can be used to infect a suitable bacterial host, and the vector, carrying a DNA insert, will be transferred into the host cell. Once inside, the ori sequence allows the cosmid to replicate as a bacterial plasmid.

9. Fig 18-11 Figure: 18-11 Caption:
A bacterial artificial chromosome (BAC). The polylinker carries a number of unique restriction sites for the insertion of foreign DNA. The arrows labeled T7 and Sp6 are promoter regions that allow expression of genes cloned between these regions.

10. Fig 18-12 Figure: 18-12 Caption:
Cloning with a plasmid vector. Plasmid vectors are isolated and cut with a restriction enzyme. The DNA to be cloned is cut with the same restriction enzyme, producing a collection of fragments. These fragments are spliced into the vector and transferred to a bacterial host for replication. Bacterial cells carrying plasmids with DNA inserts are identified by growth on selective medium and isolated. The cloned DNA is then recovered from the bacterial host for further analysis.

11. Fig 18-13 Figure: 18-13 Caption:
Cloning into a yeast artificial chromosome. The synthetic chromosome contains telomere sequences (TEL), a centromere (CEN4) derived from yeast chromosome 4, and an origin of replication (ori). These elements give the cloning vector the properties of a chromosome. TRP1, SUP4, and URA3 are yeast genes that are selectable markers for the left and right arms of the chromosome, respectively. Within the SUP4 gene is a restriction site for the enzyme SnaB1. Two BamH1 sites flank a spacer segment. Cleavage with SnaB1 and BamH1 breaks the artificial chromosome into two arms. The DNA to be cloned is treated with the same enzyme, producing a collection of fragments. The arms and fragments are ligated together, and the artificial chromosome inserted into yeast host cells. Because yeast chromosomes are large, the artificial chromosome accepts inserts in the million base pair range (Mb 5 megabase).

12. Fig 18-14 Figure: 18-14 Caption:
A Ti plasmid designed for cloning in plants. Segments of Ti DNA, including those necessary for opine synthesis and integration, are combined with bacterial segments that incorporate cloning sites and antibiotic resistance genes (kanR and tetR). The vector also contains an origin of replication (ori) and a lambda cos site that permits recovery of cloned inserts from the host plant cell.

13. Fig 18-16 Figure: 18-16_a Caption:
PCR amplification. In the polymerase chain reaction, the target DNA is denatured into single strands; each strand is then annealed to a short, complementary primer. The primers are synthetic oligonucleotides that are complementary to sequences flanking the region to be amplified. DNA polymerase and nucleotides extend the primers in the 3' direction, using the single-stranded DNA as a template. The result is a double-stranded DNA molecule with the primers incorporated into the newly synthesized strand.

14. Fig 18-16 Figure: 18-16_b Caption:
PCR amplification. In a second PCR cycle, the products of the first cycle are denatured into single strands, primers are annealed, and DNA polymerase then synthesizes new strands. Repeated cycles can amplify the original DNA sequence by more than a millionfold.

15. Fig 18-16 Figure: 18-16_c Caption:
PCR amplification. In a second PCR cycle, the products of the first cycle are denatured into single strands, primers are annealed, and DNA polymerase then synthesizes new strands. Repeated cycles can amplify the original DNA sequence by more than a millionfold.

16. Fig 18-17 Figure: 18-17 Caption:
Each human chromosome has a unique profile, resulting from the absorption of two fluorescent dyes. Based on this differential fluorescence, chromosomes can be separated from each other by flow sorting.

17. Fig 18-19 Figure: 18-19_a Caption:
Producing cDNA from mRNA. Because many eukaryotic mRNAs have a polyadenylated tail (A) of variable length at their 39-end, a short poly-dT oligonucleotide annealed to this tail serves as a primer for the enzyme reverse transcriptase. Reverse transcriptase uses the mRNA as a template to synthesize a complementary DNA strand (cDNA) and forms an mRNA/cDNA double-stranded duplex.

18. Fig 18-19 Figure: 18-19_b Caption:
Producing cDNA from mRNA. The mRNA is digested with alkali treatment, or by the enzyme RNAse H. The 39-end of the cDNA often folds back to form a hairpin loop. The loop serves as a primer for DNA polymerase, which is used to synthesize the second DNA strand. The S1 nuclease opens the hairpin loop; the result is a double-stranded cDNA molecule that can be cloned into a suitable vector, or used as a probe for library screening.

19. Fig 18-20 Figure: 18-20_a Caption:
Screening a plasmid library to recover a cloned gene. The library, present in bacteria on Petri plates, is overlaid with a DNA binding filter, and colonies are transferred to the filter. Colonies on the filter are lysed, and the DNA denatured to single strands. The filter is placed in a hybridization bag along with buffer and a labeled single-stranded DNA probe. During incubation, the probe forms a double-stranded hybrid with complementary sequences on the filter.

20. Fig 18-20 Figure: 18-20_b Caption:
Screening a plasmid library to recover a cloned gene. The filter is removed from the bag and washed to remove excess probe. Hybrids are detected by placing a piece of X-ray film over the filter and exposing it for a short time. The film is developed, and hybridization events are visualized as spots on the film. Colonies containing the insert that hybridized to the probe are identified from the orientation of the spots. Cells are picked from this colony for growth and further analysis.

21. Fig 18-21 Figure: 18-21 Caption:
In chromosome walking, the approximate location of the gene to be cloned is known. A subcloned fragment of a linked adjacent sequence recovers overlapping clones from a genomic library. This process of subcloning and probing the genomic library is repeated to recover overlapping clones until the gene in question has been reached.

22. Fig 18-23 Figure: 18-23_a Caption:
Constructing a restriction map. Samples of the 7.0-kb DNA fragments are digested with restriction enzymes: One sample is digested with HindIII, one with SalI, and one with both HindIII and SalI. The resulting fragments are separated by gel electrophoresis. The separated fragments are measured by comparing them with molecular-weight standards in an adjacent lane. Cutting the DNA with HindIII generates two fragments: 0.8 kb and 6.2 kb. Cutting with SalI produces two fragments: 1.2 kb and 5.8 kb. Models are constructed to predict the fragment sizes generated by cutting with HindIII and with SalI.

23. Fig 18-23 Figure: 18-23_b Caption:
Constructing a restriction map. Model 1 predicts that 0.4-, 0.8, and 5.8-kb fragments will result from cutting with both enzymes. Model 2 predicts that 0.8-, 1.2-, and 5.0-kb fragments will result. Comparing the predicted fragments with those observed on the gel indicates that model 1 is the correct restriction map.

24. Fig 18-24 Figure: 18-24_a Caption:
The Southern blotting technique. Samples of the DNA to be probed are cut with restriction enzymes and the fragments separated by gel electrophoresis. The pattern of fragments is visualized and photographed under ultraviolet illumination by staining the gel with ethidium bromide. The gel is then placed on a sponge wick in contact with a buffer solution and covered with a DNA-binding filter. Layers of paper towels or blotting paper are placed on top of the filter and held in place with a weight. Capillary action draws the buffer through the gel, transferring the pattern of DNA fragments from the gel to the filter.

25. Fig 18-24 Figure: 18-24_b Caption:
The Southern blotting technique. The DNA fragments on the filter are then denatured into single strands and hybridized with a labeled DNA probe. The filter is washed to remove excess probe and overlaid with a piece of X-ray film for autoradiography. The hybridized fragments show up as bands on the X-ray film.

28. Fig 18-26 Figure: 18-26_a Caption:
DNA sequencing using the chain termination method. (1) A primer is annealed to a sequence adjacent to the DNA being sequenced (usually at the insertion site of a cloning vector). (2) A reaction mixture is added to the primer–template combination. This includes DNA polymerase, the four dNTPs (one of which is radioactively labeled) and a small amount of one dideoxynucleotide. Four tubes are used, each containing a different dideoxynucleotide (ddATP, ddCTP, etc.).

29. Fig 18-26 Figure: 18-26_b Caption:
DNA sequencing using the chain termination method. (3) During primer extension, the polymerase occasionally inserts a ddNTP instead of a dNTP, terminating the synthesis of the chain, because the ddNTP does not have the 39-OH group needed to attach the next nucleotide. In the figure, ddATP and the A inserted from this didexoynucleotide are indicated with an asterisk. Over the course of the reaction, all possible termination sites will have a ddNTP inserted. (4) The newly synthesized chains are removed from the template, and the mixture is placed on a gel. Chains terminating in A are loaded in the A lane, those ending in C are loaded in the C lane, and so forth.

30. Fig 18-28 Figure: 18-28_a Caption:
In DNA sequencing using dideoxynucleotides labeled with fluorescent dyes, all four ddNTPs are added to the same tube, and during primer extension, all combinations of chains are produced. The products of the reaction are added to a single lane on a gel, and the bands are read by a detector and imaging system. This process is now automated, and robotic machines, such as those used in the Human Genome Project, sequence several hundred thousand nucleotides in a 24-hour period and then store and analyze the data automatically.

31. Fig 18-28 Figure: 18-28_b Caption:
In DNA sequencing using dideoxynucleotides labeled with fluorescent dyes, all four ddNTPs are added to the same tube, and during primer extension, all combinations of chains are produced. The products of the reaction are added to a single lane on a gel, and the bands are read by a detector and imaging system. This process is now automated, and robotic machines, such as those used in the Human Genome Project, sequence several hundred thousand nucleotides in a 24-hour period and then store and analyze the data automatically.

32. Fig 18-29 Figure: 18-29 Caption:
Automated DNA sequencing using fluorescent dyes, one for each base. The separated bases are read in order along the axis from left to right.

33. RNA
interference