1. Fig 20-1 Figure: 20-01 Caption:
Restriction fragment-length polymorphisms (RFLPs). The alleles on chromosome A and chromosome B represent DNA segments from homologous chromosomes. The region that hybridizes to a probe is shown in green. Arrows indicate the location of restriction enzyme cutting sites that define the alleles. On chromosome A, three cutting sites generate fragments of 7 kb and 3 kb. On chromosome B, only two cutting sites are present, generating a single fragment that is 10 kb in length. The absence of the cutting site in chromosome B could be the result of a single base mutation within the enzyme recognition or cutting site. Because these differences in restriction cutting sites are inherited in a codominant fashion, there are three possible genotypes: AA, AB, and BB. The allele combination carried by any individual can be detected by restriction digestion of genomic DNA (obtained, for example, from a blood sample or skin fibroblasts), followed by gel electrophoresis, transfer to a DNA binding filter, and hybridization to the appropriate probe. The fragment patterns for the three possible genotypes are shown as they would appear on a Southern blot.

2. Fig 20-2 Figure: 20-02 Caption:
Establishing linkage between a dominant trait and an RFLP allele. The pedigree shows a family with members affected by a dominant trait (filled symbols). Family members also carry two alleles of an RFLP locus assigned to a specific chromosome. A 5.0-kb allele (allele A) is present on one homolog. The B allele on the other homolog consists of two fragments (2.0 kb and 3.0 kb). The probe used in the Southern blot detects the 5-kb A allele and the 2.0-kb portion of the B allele. The RFLP pattern for each family member is shown below the appropriate pedigree symbol. Individual III-1, who is unaffected, probably received an A allele from her father and a B allele from her mother. Individual III-2 is affected and probably received an A allele from his mother and a B allele from his father. The youngest son (III-3), who is affected, received a B allele from each parent. Taken together, the pedigree and the Southern blot suggest that the mutant allele for the dominant trait and the RFLP B allele are on the same homolog and are therefore linked. Assigning a mutant allele to a chromosome by RFLP analysis is the first step in mapping a gene.

3. Fig 20-3 Figure: 20-03 Caption:
A genetic and a physical map of human chromosome 13, showing the location of markers. The genetic map for females is 203 cM, and that for males is 158 cM, reflecting the difference in recombination frequencies between females and males. When the two maps are averaged together, the result is the sex-averaged map of 178 cM shown on the left. The location of markers on the physical map is indicated by the brackets adjacent to the chromosome.

4. Fig 20-4 Figure: 20-04a Caption:
Segregation of a 2.4-kb RFLP allele with type 1 neurofibromatosis (NF1) in each of four affected offspring and their mother. This RFLP is detected by probe pA10-41, which is a DNA segment from a region near the centromere of human chromosome 17. On the basis of this result and results from tests using other probes, the locus for NF1 was assigned to chromosome 17.

5. Fig 20-4

6. Fig 20-5

7. Fig 20-6 Figure: 20-06 Caption:
The technique of amniocentesis. The position of the fetus is first determined by ultrasound, and then a needle is inserted through the abdominal and uterine wall to recover fluid and fetal cells for cytogenetic or biochemical analysis.

9. Fig 20-7 Figure: 20-07 Caption:
Diagnosis of b-thalassemia caused by a partial deletion of the b-globin gene. The family pedigree is shown above each individual’s genotype on a Southern blot. The normal b-globin allele (bA) contains three exons and two introns. The deleted b-globin allele (b0) has the third exon deleted. Arrows indicate the cutting sites for restriction enzymes used in this analysis. The normal gene produces a larger fragment (shown as the top row of fragments on the Southern blot); the smaller fragments produced by the deleted gene are represented at the bottom of the gel. The genotypes of each individual in the pedigree can be determined from the pattern of bands on the blot and is given below the blot.

10. Fig 20-8 Figure: 20-08 Caption:
Southern blot diagnosis of sickle-cell anemia. Arrows represent the location of restriction enzyme cutting sites. In the mutant  globin allele, a point mutation (GAGGTG) has destroyed a restriction enzyme cutting site, resulting in a single large fragment on a Southern blot. In the pedigree, the family has one unaffected homozygous normal daughter (II-1), an affected son (II-2), and an unaffected fetus (II-3). The genotype of each family member can be read directly from the blot and is given below the blot.

11. Fig 20-9 Figure: 20-09 Caption:
Genotype determinations, using allele-specific oligonucleotides (ASOs). In this technique, the b-globin gene is amplified by PCR, using DNA extracted from blood cells. The amplified DNA is denatured and spotted onto strips of DNA-binding filters. Each strip is hybridized to a specific ASO and visualized on X-ray film after hybridization and exposure. If all three genotypes are hybridized to an ASO from the normal b-globin allele, the pattern in (a) will be observed: AA-homozygous individuals have normal hemoglobin that has two copies of the normal b-globin gene and will show heavy hybridization; AS-heterozygous individuals carry one normal b-globin allele and one mutant allele and will show weaker hybridization; SS-homozygous sickle-cell individuals carry no normal copy of the b-globin gene and will show no hybridization to the ASO probe for the normal b-globin allele. (b) The same genotypes hybridized to the probe for the sickle-cell b-globin allele will show the reverse pattern: no hybridization by the AA genotype, weak hybridization by the heterozygote (AS), and strong hybridization by the homozygous sickle-cell genotype (SS).

12. Fig 20-10 Figure: 20-10 Caption:
Screening for cystic fibrosis (CF) by allele-specific oligonucleotides (ASOs). ASOs for the region spanning the most common mutation in CF, a three-nucleotide deletion D508, are prepared from a normal CF allele and a D508 CF allele. In screening, the CF alleles carried by an individual are amplified by PCR, using DNA extracted from blood samples, and spotted on a DNA-binding membrane. The membrane is hybridized to a mixture of the two ASOs. The genotype of each family member can be read directly from the filter. DNA from I-1 and I-2 hybridizes to both ASOs, indicating that these individuals carry a normal allele and a mutant allele and are, therefore, heterozygous. The DNA from II-1 hybridizes only to the D508 ASO, indicating that this individual is homozygous for the mutation and has cystic fibrosis. The DNA from II-2 hybridizes only to the normal ASO, indicating that this individual carries two normal alleles. II-3 has two hybridization spots and is heterozygous.

13. Fig 20-11 Figure: 20-11 Caption:
A single-stranded DNA molecule with bases extending from the sugar/phosphate backbone.

14. Fig 20-12 Figure: 20-12 Caption:
Gene screening, using a DNA microarray. (a) DNA extracted from a blood sample is amplified by PCR. In this example, primers for five genes are used, but in practice, many more are used. (b) The microarray contains single-stranded probes for the normal allele of each of the genes (Column I) and eight mutant alleles for each of the five genes (1 row = 1 gene). (c) The single-stranded PCR products are tagged with fluorescent probes and pumped into the microarray. (d) The resulting hybridization is revealed by the pattern and color of the spots on the microarray. The microarrays are scanned by a laser and analyzed by software, and the results can be presented in several formats. In practice, microarrays containing several hundred thousand probes are used. Each probe is attached to the glass substrate and occupies a different field on the microarray.

16. Fig 20-13 Figure: 20-13 Caption:
Retroviral vectors constructed from the Moloney murine leukemia virus (Moloney MLV). The native MLV genome contains a c sequence required for encapsulation and genes that encode viral coat proteins (gag), an RNA-dependent DNA polymerase (pol), and surface glycoproteins (env). At each end, the genome is flanked by long terminal repeat (LTR) sequences that control transcription and integration into the host genome. The SAX vector retains the LTR and c sequences, and it includes a bacterial neomycin resistance  gene that can be used as a selective marker. As shown, the vector carries a cloned human adenosine deaminase (hADA) gene, which is fused to an SV40 early region promoter–enhancer. The SAX construct is typical of retroviral vectors that are used in human gene therapy.

17. Fig 20-15 Figure: 20-15 Caption:
Gene therapy for treatment of severe combined immunodeficiency (SCID), a fatal disorder of the immune system caused by lack of the enzyme adenosine deaminase (ADA). The cloned human ADA gene is transferred into a viral vector, which is used to infect white blood cells removed from the patient. The transferred ADA gene is incorporated into a chromosome and becomes active. After growth to enhance their numbers, the cells are reimplanted into the patient, where they produce ADA, allowing the development of an immune response.

19. Fig 20-16 Figure: 20-16 Caption:
VNTR loci and DNA fingerprints. VNTR alleles at two loci (A and B) are shown for each individual. Arrows mark restriction enzyme cutting sites flanking the VNTRs. Restriction enzyme digestion produces a series of fragments that can be detected as bands on a Southern blot (bottom). Because of differences in the number of repeats at each locus differ, the overall pattern of bands is distinct for each individual, even though one band is shared (the B2 allele band). Such a pattern is known as a DNA fingerprint.

20. Fig 20-17

21. Figure: UN
Caption:
DNA fingerprinting of mother (M), putative father (F) and child (C)

22. Figure: UN
Caption:
DNA fingerprinting of mother (M), putative father (F) and child (C)

23. Ethical, Legal, and Social Implications
(ELSI) Program

24. Figure: UN
Caption:
DNA obtained from family members' white blood cells and cut with HindIII

25. Figure: UN
Caption:
Pedigree of a rare disease state

26. Figure: UN
Caption:
DNA samples from generations II and II analyzed and identified by Probe A and B

27. Figure: UN
Caption:
Pedigree and DNA blot from a fetus