Molecular Tools in Cancer Research
Summary of Key Points
• Our understanding and treatment of cancer have always relied heavily on parallel developments in biological research. Molecular biology provides the basic tools to study genes involved with cancer growth patterns and tumor suppression. An advanced understanding of the molecular processes governing cell growth and differentiation has revolutionized the diagnosis and prognosis of malignant disorders.
• This introductory chapter relates basic principles of molecular biology to emerging perspectives on the origin and progression of cancer and explains newly developed laboratory techniques, including whole genome analysis, expression profiling, and refined genetic manipulation in animal models, thus providing the conceptual and technical background necessary to grasp the central principles and new methods of current cancer research.
1. It would be most appropriate to use which technique to study and identify cancer mechanisms that display different levels of alternative spliced messenger ribonucleic acids (mRNAs)?
2. The allelic variance in the form of single-nucleotide polymorphisms (SNPs) encountered in the human population can be used in cancer biology because
A they mark variability in the coding sequence that leads to diseased phenotypes and cancer prognosis.
B they provide integration of individual characteristics and disease.
C they allow identification of specific allelic combinations that can be associated with disease.
3. How do epigenetic changes differ from changes in the genetic code?
B Epigenetic changes are not transmitted to daughter cells.
C Epigenetic changes are heritable traits associated with dynamic changes in chromatin structure, regulating transcription.
D They are mutations in noncoding DNA sequences that affect transcription of important regulatory genes.
E Because they affect the third base of the codon, they do not affect the final protein identity.
4. When referring to transcriptomes, proteomes, and interactomes which statement is INCORRECT?
A They are all associated with analysis of global biological functions in cells or organisms.
B The transcriptome is the total RNA profile, the proteome is the analysis of all proteins expressed, and the interactome is the analysis of interactions between cellular molecules.
C RNA-seq and microarray are techniques associated with transcriptome analysis.
D These are some of the components used for the global “omic” analysis of individuals (iHOP).
E Cellular proteomics must reflect exactly what is observed in transcriptome analysis, thus working as a validation of the “strength” of the study.
5. Mouse transgenics models are characterized
A by the use of homologous recombination at specific genomic targets, differing from knockout/knockin models that use random integration of DNA fragments.
B by the use of the endogenous regulatory regions.
C as the best model to analyze gain of function and dominant negative effects commonly seen in cancers.
D by the ability to be genetically coupled with the use of Cre-mediated site-specific recombination and thus allow temporal and spatial control expression of the tested gene.
E by the use of DNA electroporation in embryonic stem cells, followed by negative and positive selection of genomic integration.
1. Answer: C. RNA-seq allows analysis of both transcriptional levels and the presence of alternatively expressed isoforms. Microarray allows differential analysis of transcripts but cannot detect variations associated with differential splicing. PCR is a technique associated with analysis and identification of single targets. ChIP-seq is a technique for global analysis of promoter occupancy at regulatory sequences in the deoxyribonucleic acid (DNA), and Y2H is used for the study of protein interactions.
2. Answer: E. A is wrong because it is not necessarily associated with coding sequences, and D is incorrect because they represent variations that exist between individuals that MIGHT be associated with disease.
3. Answer: C. A is wrong because the genetic code relates to the primary sequence of the DNA (combination of A, T, C, and G) bases that is transmitted by DNA replication and controls cellular function by mRNA-protein expression. B is not correct because epigenetic changes can be transmitted and in fact are essential for cell fate and regulatory events at the cellular level. D is wrong because it describes mutations in promoter and regulatory regions (genetic code). E is incorrect because it is the description of an event associated with the reading of the genetic code, where changes at the third base of the codon sometimes translates to the same amino acid (silent mutations).
4. Answer: E. Those techniques are complementary, representing different stages of the information transfer that occurs in cells and is essential for its normal function.
5. Answer: C. All the others are characteristics of knockout/knockin models. Transgenics rely on the direction injection of modified DNA that contains regulatory regions controlling the gene of interest. Control can be obtained through the use of inducible promoters. Knockout/knockin models use homologous recombination driven by arms surrounding the region of interest. To select the correct recombined clones, ES electroporated cells are selected with positive selection (to confirm integration of cassette) and negative selection (to confirm correct position in the genome). In addition to the obvious advantage of having the endogenous control regions, the knockout/knockin strategy allows also integration of recombination sites that can be used together with mice expressing specific recombinases, thus allowing temporal and spatial control of modification.
