Cystic fibrosis, a genetic disease, can be diagnosed before birth and carriers can be identified. It is caused by mutations in the gene responsible for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation involves a three-base deletion that leads to the loss of a phenylalanine residue from the CFTR protein. This deletion results in a shorter mutant allele compared to the normal allele. To distinguish between the two alleles, a PCR test can be conducted by amplifying that specific region of the DNA and examining the size of the PCR products. Homozygous normal individuals, carriers (heterozygous), and affected individuals (homozygous mutant) can be differentiated using this PCR test. 

Multiplex PCR, the simultaneous amplification of multiple regions of a target DNA using multiple primer pairs, is employed for genes with multiple exons, such as the CFTR gene with its 27 exons. 

Biotechnology tools not only facilitate the study of gene structure but also enable the analysis of mRNA and protein products resulting from gene expression. mRNA levels can be determined through various methods, including hybridization of labeled probes to either mRNA or cDNA produced from mRNA using reverse transcriptase PCR (RT-PCR). Northern blots are used to separate and identify mRNA molecules by electrophoresis, followed by transfer to a membrane and hybridization with radiolabeled probes. The resulting bands on the autoradiography image provide information about the amount and size of the mRNA molecules in the sample.

DNA microarrays, consisting of thousands of immobilized ssDNA sequences, are utilized to analyze genomic DNA for gene variations or mutations (genotyping) and to examine mRNA production patterns (gene expression analysis). By converting the mRNA population from a specific cell type into cDNA and labeling it with a fluorescent tag, researchers can determine the amount of that particular mRNA in the sample using a gene chip. Each spot on the chip corresponds to a different gene, allowing simultaneous analysis of thousands of genes. DNA microarrays are useful for studying gene expression patterns in different cell types and can be used for subclassifying cancers, like breast cancer, to optimize treatment.

Protein analysis involves investigating the types and amounts of proteins in cells. However, protein abundance does not always directly correlate with mRNA levels due to variations in translation efficiency and posttranslational modifications. When studying a large number of proteins, automated methods employing techniques such as mass spectrometry and two-dimensional electrophoresis are used. Labeled antibodies are employed to detect and quantify specific proteins and identify posttranslational modifications when investigating one or a limited number of proteins.
Prenatal diagnosis and carrier detection of cystic fibrosis: Cystic fibrosis is an autosomal-recessive genetic disease resulting from mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. The most common mutation is a three-base deletion that results in the loss of a phenylalanine residue from the CFTR protein (see p. 450). Because the mutant allele is three bases shorter than the normal allele, it is possible to distinguish them from each other by the size of the PCR products obtained by amplifying that portion of the DNA. Figure 34.22 illustrates how the results of such a PCR test can distinguish between homozygous normal, heterozygous (carriers), and homozygous mutant (affected) individuals.
The simultaneous amplification of multiple regions of a target DNA using multiple primer pairs is known as multiplex PCR. It allows detection of the loss of ≥1 exons in a gene with many exons such as the gene for CFTR, which has 27 exons.
VIII. GENE EXPRESSION ANALYSIS
The tools of biotechnology not only allow the study of gene structure, but also provide ways of analyzing the mRNA and protein products of gene expression.
A. Determining messenger RNA levels mRNA levels are usually determined by the hybridization of labeled probes to either mRNA itself or to cDNA produced from mRNA. [Note: Amplification by PCR of cDNA made from mRNA by retroviral reverse transcriptase (RT) is referred to as RT-PCR.] 1. Northern blots
Northern blots are similar to Southern blots (see Fig. 34.13), except that the sample contains a mixture of mRNA molecules that are separated by electrophoresis, then transferred to a membrane and hybridized with a radiolabeled probe. The bands obtained by autoradiography give a measure of the amount and size of the mRNA molecules in the sample.
2. Microarrays: DNA microarrays contain thousands of immobilized ssDNA sequences organized in an area no larger than a microscope slide. These microarrays are used to analyze a sample for the presence of gene variations or mutations (genotyping) or to determine the patterns of mRNA production (gene expression analysis), analyzing thousands of genes at the same time. For genotyping analysis, the sample is from genomic DNA. For expression analysis, the population of mRNA molecules from a particular cell type is converted to cDNA and labeled with a fluorescent tag (Fig. 34.23). This mixture is then exposed to a gene (or, DNA) chip, which is a glass slide or membrane containing thousands of tiny spots of DNA, each corresponding to a different gene. The amount of fluorescence bound to each spot is a measure of the amount of that particular mRNA in the sample. DNA microarrays are used to determine the differing patterns of gene expression in two different types of cell (for example, normal and cancer cells; see Fig. 34.23). They can also be used to subclassify cancers, such as breast cancer, to optimize treatment. [Note: Microarrays involving proteins and the antibodies or other proteins that recognize them are being used to identify biomarkers to aid in the diagnosis, prognosis, and treatment of disease based on a patient’s protein expression profile. Protein (and DNA) microarrays are important tools in the development of personalized (precision) medicine in which the treatment and/or prevention strategies consider the genetic, environmental, and lifestyle variations among individuals.]
B. Protein analysis
The kinds and amounts of proteins in cells do not always directly correspond to the amounts of mRNA present. Some mRNA are translated more efficiently than others, and some proteins undergo posttranslational modification. When analyzing the abundance and interactions of a large number of proteins, automated methods involving a variety of techniques, such as mass spectrometry and two-dimensional electrophoresis, are used. When investigating one, or a limited number of proteins, labeled antibodies (Ab) are used to detect and quantify specific proteins and to determine posttranslational modifications.
1.