Wallach's Interpretation of Diagnostic Tests: Pathways to Arriving at a Clinical Diagnosis (458 page)

Suggested Readings

Levilliers J, et al. Exchange of terminal portions of X- and Y-short arms in human XY females.
Proc Natl Acad Sci U S A.
1989;86:2296–3000.

Therman E, Susman B. The similarity of phenotypic effects caused by Xp and Xq deletions in the human female: a hypothesis.
Hum Genet.
1990;85:175–183.

   
GLOSSARY FOR MOLECULAR METHODS TERMINOLOGY

Array comparative genomic hybridization (aCGH)
: A microarray-based technique to detect abnormalities in DNA copy number (i.e., missing or extra pieces of chromosomes) that can detect smaller abnormalities than a standard chromosomal analysis but will not detect balanced chromosomal rearrangements, such as translocations. It is used as an adjunct or replacement to chromosome analysis; does not detect single-gene mutations.

Allele-specific oligonucleotide (ASO) testing
: Detection of a specific mutation using a synthetic segment of DNA approximately 20 base pairs in length (an oligonucleotide) that binds to and hence identifies the complementary sequence in a DNA sample.

bDNA testing
: Branched DNA testing is a test in which a phosphorescent chemical that is known to bind to RNA is added to the suspect DNA. The more brightly the test sample glows, the greater amount of RNA that is present in the sample; this test is used to directly measure the amount of RNA in a sample (e.g., viral load).

Bead array
: Array consisting of addressable beads either impregnated with different concentrations of fluorescent dye or labeled with some type of bar code. The addressable beads enable the identification of specific oligonucleotide binding to the bead’s surface. The combination of specific oligonucleotide bound to a specific bead is decoded to determine the presence or absence of a particular target DNA sequence.

Chromosome analysis
: Provides an overview of the genome through microscopic visual inspection of banded mitotic chromosomes. It requires cells to be in metaphase; therefore, cells must be
cultured
and chemically arrested in metaphase to obtain chromosomes that can be visualized. Aberrations must be at least 5–10 Mb to be appreciated.

Denaturing gradient gel electrophoresis (DGGE)
: Detects changes in DNA sequence based on differences in energy required for separation during electrophoresis of double-stranded DNA fragments of the same size into single-stranded DNA on a polyacrylamide gel with gradient of denaturant (chemical denaturants as formamide and urea) at elevated temperatures. DNA fragments are progressing through the gel according to their melting (denaturing) temperature, which is dependent on the ratio of GC to AT base pairs that make up a particular segment of DNA. A confirmatory test is required for mutation analysis.

Denaturing high-performance liquid chromatography (DHPLC)
—a large-scale chromatographic method used for identification of sequence variation allows rapid detection of mutations by heteroduplex formation between wild-type and mutant DNA. Exon sequencing is required to characterize the mutation.

Diagnostic test
: Test performed to confirm the presence of a specific medical condition. Molecular tests are used currently as an aid in evaluation of patients suspected of/with infectious diseases, genetic disorders, and other disorders where there are established known genetic risk factors. Also in the last few years, pharmacogenetic testing has evolved, creating personalized approached to drug choices and dosing based on the individual’s variants.

Fluorescence in situ hybridization (FISH)
: Molecular hybridization of a fluorescently labeled, cloned sequence to a mitotic chromosome or to an interphase nucleus. FISH is used to interrogate a specific region of the genome, designed to detect chromosome rearrangements or aberrations that are least 100 kb in size.

Fluorescence-based fragment size analysis
: A method for detection of mutations/ variants that cause change in the size of a DNA fragment, such as expansion or contraction of tandem repeats. The size of fluorescently labeled fragments, amplified by PCR, is detected using the capillary electrophoresis and then interpreted using the analysis software. Multiple-colored fluorescent dyes can be detected in one sample. One of the dye colors is used for a labeled size standard that is added to each lane. The analysis software uses the size standard to create a standard curve for each lane and then determines the length of each dye-labeled fragment by comparing it with the standard curve for that specific lane.

Fluorescence resonance energy transfer (FRET)
: Mechanism describing energy transfer between two chromophores.

Genome
: Complete DNA sequence, containing the entire genetic information, of a gamete, an individual, a population, or a species.

Genomics
: Field of genetics concerned with structural and functional studies of the genome.

Genotyping
: Process of determining the genetic makeup of an individual, usually with methods such as PCR, DNA sequencing, ASO probes, and hybridization to DNA microarrays or beads.

Haplotype analysis
: Determination of the extent of association to a trait of a set of closely linked loci such as a group of genes that occupy a specific position on one chromosome that tend to be inherited together.

Hybridization
: Used to determine the degree of sequence identity, as well as specific sequences between nucleic acids by interacting single-stranded DNA or RNA in solution or with one component immobilized so that complexes called hybrids are formed by molecules with similar, complementary sequences.

Invader chemistry
: Composed of two simultaneous isothermal reactions, a primary reaction that detects mutation and secondary reaction that amplifies the signal. The fluorescent signal is generated by the cleavage of a synthetic oligonucleotide probe labeled with FRET.

Karyotype
: Ordered pairing of chromosomes that aids in detecting abnormalities.

Ligase chain reaction (LCR)
: DNA amplification technology based on the ligation of two pairs of synthetic oligonucleotides, which hybridize at adjacent positions to complementary strands of a target DNA.

Linkage analysis
: Testing DNA sequence polymorphisms (normal variants) that are near or within a gene of interest to track inheritance of a disease-causing mutation.

Microarray
: Consists of the hybridization of a nucleic acid sample (target) to a very large set of oligonucleotide probes, which are attached to a solid support or in solution, to determine sequence, or to detect variations in a gene sequence or expression or for gene mapping.

Multiplex ligation
–dependent probe amplification (MLPA): Detects deletions and duplications, determines the copy number of all or selected exons within a gene with high sensitivity.

Mutation scanning
: A search for novel sequence variants within a specific DNA fragment.

Next-generation sequencing
(Next Gen, NGS): The bases of DNA fragments are sequentially identified from signals emitted as each fragment is resynthesized from a DNA template across millions of reactions in a massively parallel fashion; multiple, fragmented sequence reads are assembled together on the basis of their overlapping location. This advance enables rapid sequencing of large stretches of DNA base pairs spanning entire genomes.

   
The automated Sanger methodology
is referred to as a “first-generation technology,” and NGS technologies are essentially grouped into second-generation (2G) and third-generation (3G) approaches. Several 2G approaches are commercially available (e.g., Roche-454, Illumina-Solexa, Applied Biosystems-SOLiD). The third-generation (3G) platforms are represented by the Helicos HeliScope, the Pacific Bioscience, and Oxford Nanopore Technologies.

   
Second-generation (2G) platforms
use either “emulsion PCR” (Roche-454, Applied Biosystems-SOLiD) or “bridge PCR” (Illumina) for target amplification, followed by cyclic-array sequencing, the sequencing of DNA on a dense array, for example, streptavidin beads (Roche-454), flow cells (Illumina), or glass surfaces (Applied Biosystems-SOLiD) by alternating cycles of enzymedriven biochemistry and imaging-based data collection. All 2G technologies are engineered to obtain massively parallel output.

   
3G technologies
use a single molecule template approach, do not use PCR amplification step, and avoid the cyclic-array approach and thereby enable further massive parallelization. These methods (Read-out) include differential conductance across nanopores (Oxford Nanopore Technology) and single molecule real-time sequencing using Fluorescence Resonance Energy Transfer (Applied Biosystems) or zero-mode waveguide detectors (Pacific Biosciences).

Current applications of NGS include de novo sequencing, resequencing, epigenetics, and metagenomics.

Non-Invasive Prenatal Testing (NIPT)
: Cell-free fetal DNA circulating in maternal blood, is analyzed for trisomy 21 and other fetal chromosomal aneuploidies.

Northern blot
: Used to study gene expression by detection of RNA with a hybridization probe complementary to part of or an entire size-separated RNA sample.

Oligonucleotide ligation assay (OLA)
: Rapid, sensitive, and specific method for the detection of known SNPs that is based on the joining of two adjacent oligonucleotide probes (capture and reporter oligos) using a DNA ligase while they are annealed to a complementary DNA target. The detection of an SNP occurs by the ability of DNA ligase to join probes that are perfectly matched to a complementary target sequence, whereas a 3′ mismatch in the capture probe prevents ligation.

Polymerase chain reaction (PCR)
: Molecular technique by which a short DNA (or RNA following reverse transcription) sequence is amplified by two flanking oligonucleotide primers used in repeated cycles of primer extension and DNA synthesis with DNA polymerase.