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Molecular Methods

John W. Belmont

Molecular analysis of genes and genomes can be central to arriving of groups of tissue-specific genes primarily encode transcription
at a precision diagnosis. This chapter reviews the basic principles factors that regulate the rate of messenger RNA (mRNA) tran-
that underlie clinical molecular genetic testing, assesses representa- scription on their target genes. A few of these apparently act as
tive standard methods that are widely employed, describes new “master” genes during particular developmental processes or in
DNA sequencing methods that are being rapidly introduced into specific cell lineages. Some of these master genes (e.g., PAX5),
clinical diagnostic laboratories, and suggests a multidisciplinary may be involved in leukemias and lymphomas. So far, immu-
approach for implementation in immunological disorders. nodeficiency genes appear to be in the category of genes that
involve either innate or adaptive immunity controlling cell growth,
BASIC PRINCIPLES differentiation, effector functions, or apoptosis. 1,2
Genome Structure and Gene Expression Polymorphic Variation and Linkage
A genetic map relates one gene to another based on how often
KEY CONCEPTS they are inherited together. Within a specific region of the DNA,
Human Genomics the maternal and paternal copies of the genome may be nonidenti-
cal, and the variants are called alleles. Protein and nucleotide
• The human genome encompasses approximately 20 000 protein-coding differences are called polymorphisms when they are frequent
genes, but each cell expresses only a subset of those genes. enough to be found in 1–5% of the general population. Variations
• Genetic and physical maps of the genome are essential to molecular
diagnosis of immune system diseases. in single nucleotides occur in ≈1/100 bases when whole-genome
3
• Genetic maps depend on the coinheritance of DNA segments—linkage— sequencing is used to survey individuals. Polymorphisms arise
to associate DNA variants with disease. over time in a group of individuals because of mutations in the
• Physical maps of the genome describe the exact gene locations on DNA (Fig. 96.1). Some polymorphisms involve simple sequence
a chromosome. The genome DNA sequence is the finest scale physical repeats so that there is variation in the number of repeat units.
map of the genome. These are called short tandem repeats (STRs) and are widely used
in forensic identification. In contrast to STRs, the most common
The human genome is thought to have about 20 000 protein- polymorphisms involve a change in a single nucleotide position.
coding genes distributed on 23 pairs of chromosomes (http:// Large databases of single nucleotide polymorphisms (SNPs,
www.ncbi.nlm.nih.gov/entrez/). The total length of one copy of pronounced “snips”) have been accumulated (http://www.ncbi
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the genome is ≈3 × 10 nucleotide bases. The protein-coding .nlm.nih.gov/SNP/). It is thought that there could be >30 × 10
segments (exons) are split by noncoding DNA sequences (introns). SNPs with minor allele frequency >5% available across the human
The aggregate protein coding sequences, referred to as the “exome,” genome. Many SNPs are found in noncoding DNA, and these
account for about 1–1.5% of the genome. Some of the remaining have been shown to play a significant, albeit incompletely
DNA contains regulatory elements that direct the expression of understood, role in common human diseases.
genes, control chromatin conformation, encode regulatory RNAs, The composition of alleles within genetic loci in an individual
act as origins for DNA replication, and participate in three- is called the genotype. The individual’s genotype interacts with
dimensional (3D) looping to produce the large-scale chromosome the environment throughout life to create the phenotype. Some
structure. About 40% of the total DNA is accounted for by families components of the phenotype, such as body weight, are simple
of repeated sequences. These repeat elements are generally silent, to measure, whereas other clinically important phenotypes are
but they may be involved in some types of gene regulation and based on complex laboratory evaluation (e.g., in T-cell prolifera-
can become involved in mutational mechanisms of deletion, tion). A key distinction is drawn between discrete traits (normal
duplication, and insertion. Each cell expresses only a subset of vs abnormal) and quantitative traits (continuous range of values).
the entire gene repertoire. “Housekeeping” genes are expressed Polymorphisms account for some of the variation observed at
in almost all tissues and cell types, where they perform basic the phenotype level between healthy individuals or between
metabolic and structural functions. Other genes are under very populations, and the cumulative percentage of the variation
specific control, and their expression is restricted to one or a explained by genetic variation is called heritability. In monogenic
few cell types. Differential gene expression specifies the unique diseases (also called mendelian diseases), the presence of
functions of cells (e.g., immunoglobulin [Ig] in B cells, and T-cell mutation(s) is usually considered necessary and sufficient to
receptor [TCR] in T cells). Genes that coordinate the expression cause disease. The landscape of genetic variation in each individual

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