Molecular Biotechnology: Principles and Applications - Glick B., Pasternak J. 2002

Molecular Biotechnology of Microbial Systems
Molecular Diagnostics
Molecular Diagnostics of Genetic Diseases

Diagnosing specific hereditary human diseases at the genetic level determines whether the tested individuals or their offspring fall into a high-risk genetic group. DNA analysis can be used to identify carriers of hereditary disease genes, as well as for prenatal and presymptomatic Diagnosis of serious Genetic Disorders.

DNA-level tests allow for the precise detection of specific Mutations. Previously, Biochemical Methods based on detecting the product of the analyzed Gene were used for this purpose. DNA tests do not require expression of the mutant gene to detect it, enabling The Development of screening systems for all Monogenic Diseases.

Sickle Cell anemia Sickle cell anemia is a genetic disease caused by a single nucleotide substitution in the codon corresponding to the sixth amino acid in the ß-chain of the Hemoglobin molecule. In individuals homozygous for the mutant gene (S/S), red Blood Cells have an unusual sickle shape; this is due to a conformational distortion of the hemoglobin molecule resulting from the substitution of glutamic acid with valine. Mutant hemoglobin cannot transport oxygen efficiently, and these patients develop severe anemia with progressive damage to The Heart, Lungs, Brain, joints, and other Organs. In individuals heterozygous for this gene (A/S) (carriers of the genetic trait), red blood cells have a normal shape, and symptoms appear only under extreme conditions (at high altitudes or at extremely high or low temperatures, when oxygen supply to the body is reduced). If both parents are heterozygous (genotype A/S), the probability that their child will be homozygous for the mutant gene (S/S) (i.e., will have sickle cell anemia) is 25%. The sickle cell gene occurs with high frequency among African Americans and their descendants, as well as among Hispanics. In the United States, screening is conducted to identify carriers of the sickle cell gene who might pass it on to their offspring. Let us consider one of the tests used for this purpose.

The single nucleotide substitution in the ß-globin gene that leads to sickle cell anemia is accompanied by the elimination of a restriction site for the endonuclease CvnI. This enzyme recognizes the sequence CCTNAGG and cleaves the DNA molecule between the C and T bases (where N is any of the four NUCLEOTIDES). In the normal gene, this sequence is CCTGAGG, whereas in the sickle cell gene, it is CCTGTGG. DNA diagnosis of this disease is based on this difference (Fig. 9.9).

Using primers flanking the CvnI site, a small amount of the test DNA is amplified by PCR (Fig. 9.9, A). The amplified fragment is digested with CvnI, and the restriction products are separated by gel Electrophoresis and stained with ethidium bromide. In the presence of the CvnI site, a specific band pattern appears on the electropherogram (Fig. 9.9, C), which differs from that in the absence of the CvnI site. This method allows for an easy and rapid Determination of the subject's genetic status without performing a Hybridization Procedure.

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Fig. 9.9. Detection of the mutant gene responsible for the development of sickle cell anemia. A. PCR Amplification of a region of the ß-globin gene containing sites for the endonuclease CvnI, one of which is absent in the mutant gene. B. Restriction Digestion of the resulting PCR products with CvnI. The normal gene contains three CvnI sites in the DNA segment flanked by the primers, while the mutant gene contains two. C. Electrophoretic Separation of fragments obtained by digesting PCR-amplified ß-globin DNA with CvnI. AA — homozygosity for the normal ß-globin gene, AS — heterozygosity, SS — homozygosity for the sickle cell gene.

The PCR/OLA Method

Not all genetic disorders resulting in defective genes are accompanied by the loss or alteration of restriction sites; therefore, other approaches are used to detect single-nucleotide substitutions. One of these combines PCR with the oligonucleotide ligation assay (OLA), referred to as PCR/OLA.

Suppose that a normal gene has an A•T pair at a specific site (say, position 106), while the mutant gene has a G•C pair at the same site. Knowing The nucleotide sequences flanking the 106th nucleotide, one can synthesize two short (20-nucleotide) fragments adjacent to this site and complementary to opposite strands (Fig. 9.10). The key feature of this pair of oligonucleotides is that the 3'-terminal nucleotide of one (probe X) is complementary to the base at position 106 of the normal sequence, while the 5'-terminal nucleotide of the second (probe Y) is complementary to the nucleotide adjacent to the 106th nucleotide. When these probes are annealed to a target DNA containing the normal sequence (amplified by PCR), they hybridize completely, and upon addition of DNA ligase to the reaction mixture, probes X and Y are covalently joined. However, if these probes anneal to mutant DNA containing a Substitution at the 106th nucleotide, the non-complementary 3'-terminal nucleotide of probe X cannot pair with it. Although probe Y still hybridizes completely, DNA ligase cannot join probes X and Y.

Fig. 9.10. PCR/OLA method. B — biotin; D — digoxigenin; AP — alkaline phosphatase; SA — streptavidin.

Other oligonucleotide probes can be synthesized to perfectly match the sequence with the mutant 106th nucleotide. With such a set of probes, ligation will occur when they anneal to the mutant DNA target, but not when they anneal to the normal target. Thus, the PCR/OLA method distinguishes between two scenarios: probe ligation and the absence of ligation.

To determine whether ligation has occurred, the 5' end of probe X is labeled with biotin, and the 3' end of probe Y is labeled with digoxigenin, a low-molecular-weight compound that binds to a specific antibody. Following hybridization and ligation, the DNA is denatured to release the hybridized probe, and the mixture is transferred to a small streptavidin-coated plastic well. The well is washed to remove all material except for the biotinylated probe bound to streptavidin. Next, anti-digoxigenin Antibodies conjugated to alkaline phosphatase are added to the well. After a wash step to remove unbound conjugate, a colorless chromogenic substrate is added. Development of color in the well indicates binding of the anti-digoxigenin antibody to the digoxigenin-labeled probe, meaning that this probe was ligated to the biotin-labeled probe. If no color develops, ligation did not occur.

Using two pairs of probes, the genetic status of any individual can be determined. For example, DNA from heterozygous carriers yields a positive result with both probe pairs; DNA from individuals with two copies of the normal gene reacts only with the probe set containing the nucleotide complementary to the normal site; and finally, DNA from individuals with two altered copies of the gene reacts only with the probe set detecting the mutant site. To minimize The amount of starting DNA required for the assay, the region of the target DNA containing the site of interest is amplified by PCR prior to hybridization.

PCR/OLA is a rapid, sensitive, and highly specific method. All of its steps can be automated, allowing up to 1,200 tests to be performed per day.

A simpler, though less sensitive, variant of PCR/OLA is the ligase chain reaction (LCR). The test DNA is mixed with an excess of the two indicator probes described above in the presence of a thermostable DNA ligase. Ligation is carried out at 65 °C, then the Temperature is raised to 94 °C to denature the resulting probe-target hybrids, and then lowered back to 65 °C to allow free, unligated indicator OLA probes to hybridize with the target DNA. This cycle is repeated 20 times. If the indicator OLA probes are fully complementary to the target DNA, ligation will occur in each cycle, and after 20 cycles, enough ligation products (joined X and Y probes) will accumulate to be detected by electrophoresis or ELISA. If the complementarity is incomplete, ligation will not occur, and no products will be detected.

Genotyping using fluorescently labeled PCR primers Colorimetric genotyping is based on The Use of PCR primers labeled with different fluorescent Dyes. To distinguish between mutant and wild-type DNA, PCR is performed with two different primers. One of them (P1) is complementary to the wild-type DNA and is labeled at the 5' end with rhodamine (red), while the other (P3) is complementary to the mutant DNA and is labeled at the 5' end with fluorescein (green) (Fig. 9.11). In both cases, amplification is carried out in the presence of a third, unlabeled primer (P2) complementary to the opposite strand. Since PCR can only proceed if the primer is fully complementary to the target DNA, in the presence of all three primers in the reaction mixture, either wild-type DNA, mutant DNA, or both will be amplified, depending on the template DNA. If an individual is homozygous for wild-type DNA, red fluorescence will be observed after PCR and removal of excess primers; if homozygous for mutant DNA, green fluorescence will be observed; and if both mutant and wild-type DNA are present (i.e., the individual is heterozygous), yellow fluorescence will be observed. This method can be automated and adapted for any single-nucleotide target site in any gene with a known nucleotide sequence.

Mutations at different sites of a single gene

By no means are all genetic diseases caused by a single specific change in a gene. In most cases, mutations occur at different sites within the same gene but lead to the same genetic disorder. An example is ß-thalassemia, a hereditary disease associated with the loss of ß-globin activity. Heterozygous carriers usually present with mild anemia. However, individuals homozygous for any of at least eight possible mutant sites require regular blood transfusions and other treatments to survive. Since a mutation at any of the eight specific sites of the ß-globin gene can lead to ß-thalassemia, at least eight different tests must be performed. Such diagnosis is possible, though highly expensive.

Fig. 9.11. Detection of a point mutation using fluorescently labeled PCR primers. A. Using primers P1 and P2, wild-type DNA is amplified. Mutant DNA is not amplified with these primers due to a mismatch with primer P1. The 5' end of primer P1 is labeled with rhodamine, and primer P2 is unlabeled. B. Using primers P3 and P2, mutant DNA is amplified; wild-type DNA is not amplified in this case. The 5' end of primer P3 is labeled with fluorescein, and primer P2 is unlabeled. The '+' and '—' signs correspond to the wild-type and mutant sites, respectively. For genotypes '+/+', '+/—', and '—/—', PCR products containing only rhodamine, a mixture of rhodamine and fluorescein, and only fluorescein are formed, resulting in red, yellow, and green fluorescence, respectively.

Therefore, to screen for mutations occurring at different sites of a single gene, a single-reaction PCR/hybridization strategy was developed. For this purpose, a set of specific 20-nucleotide probes is synthesized, each fully complementary to a fragment of the target gene carrying a known mutation. A poly(dT) homopolymer of approximately 400 nucleotides is attached to the 3' end of each probe, anchoring the DNA probe to a pre-marked spot on a nylon filter, while the rest of the probe remains free to hybridize (Fig. 9.12). The segments of the test DNA, each containing one of the possible mutation sites, are simultaneously amplified by PCR, with one primer of each pair labeled with biotin at the 5' end. The amplified target DNA fragments are hybridized with the filter-bound probes under conditions that ensure hybridization of only fully complementary sequences. Streptavidin conjugated to alkaline phosphatase (horseradish peroxidase or urease can also be used) is added to the hybridization mixture. After hybridization, the filter is washed, and a colorless substrate is added. If There is a perfect match between the amplified target DNA segment and a specific oligonucleotide probe, a colored spot will appear on the filter. Multiple spots corresponding to a variety of different specific oligonucleotide probes can be applied to the same filter. By analyzing this color pattern, one of many possible mutation sites can be identified.

Fig. 9.12. Detection of mutations at different sites of a single gene. B — biotin; SA — streptavidin; AP — alkaline phosphatase.