Drug Delivery and Biopharmaceuticals in Oncology II Flashcards
(17 cards)
Define RT-PCR
(Reverse Transcriptase PCR): Uses mRNA as a template, converts it to cDNA.
Define qPCR
(Quantitative PCR): Provides quantification of template using fluorescent probes. Two fluorescent TaqMan probes - One specific for wild-type sequence the other for the mutation
Unlabelled forward and reverse primers
What do mutations affect?
Improve efficacy of the drug
Decrease efficacy of the drug
What is a point mutation?
Single nucleotide changes affecting one biochemical function.
Detected using PCR.
What is a single nucleotide polymorphism?
Single base change in the DNA sequence that is present in at least 1% of the population.
Location: Can occur in coding (exons) or non-coding (introns, promoters, intergenic regions) of the genome.
Some SNPs have no effect on protein function (silent mutations).
Others can alter protein function or expression, potentially leading to disease (e.g., sickle cell anemia, cystic fibrosis).
Some can influence drug metabolism, affecting drug efficacy and toxicity (pharmacogenomics).
Where have mutations been found in the EGFR gene?
Exons 18,19 and 21 which encode for the intracellular TK domain
How can PCR be used to detect mutations?
PCR can be used to detect these changes by designing primers to give different sized PCR products in the absence and presence of the mutation
Key to know what mutation the patient has
Name the two mutations which result in a change in the restriction site
G719X and L858R mutations - this means that the restriction enzyme can no longer recognise the restriction site when this mutation is present
Exon 19 deletion gives an amplicon with a different size compared to the wild type
Define allele-specific PCR (ASPCR)
Can detect point mutations without relying on changes in restriction sites
Relies on the fact that the correct base at the 3’ end of a primer is crucial for DNA synthesis
Uses fluorescent probes for mutation-specific detection
Define microarrays
Are microchips or flow cells containing many microscopic spots of different DNA or RNA probes
Used to assess expression of large numbers genes, determine genotype or sequence from the test sample
Can determine whether a particular gene is downregulated or upregulated in a particular disease state
identify different drug targets by looking at mechanisms of disease
What are the applications that microarray technologies are used in?
Basic research, including target identification
Human diagnostics
Personalised medicines - what mutations a patient has?
How is microarray technologies - gene expression carried out?
1) sample preparation - extract mRNA from cells, convert to cDNA and label with florescent dyes
2) hybridisation - mix with labelled cDNA with microarray chip containing probes, hybridise under controlled conditions, will bind to the sequence, complimentary sequence if its present on the chip
3) measure fluorescent signals to quantify gene expression, analyse data to identify differentially expressed genes
Describe gene expression analysis
Allows comparison of gene activity across different cell types, conditions or disease states
Commonly used to differentiate cancer cell lines from normal cells
For example, breast cancer studies have identified distinct gene expression patterns:
Group A: Downregulated in all samples.
Group B: Downregulated in control cells but upregulated in others.
Group C: Upregulated in all samples.
Group D: Upregulated in control cells and downregulated in others.
Define Affymetrix gene-chip
High-density SNP and copy number variation probes.
Examples include the Genome-Wide Human SNP 6.0 array.
Define next-generation sequencing (NGS)
Define ONT
DNA passes through a protein nanopore embedded in a membrane
as each nucleotide moves through a pore it disrupts the ionic current creating a unique signal
Define Oxford Nanopore Technologies
Real-time DNA and RNA sequencing, known for its ability to produce ultra-long reads and detect base modifications directly. It uses a unique method called nanopore sequencing, which allows DNA to be read as it passes through a microscopic protein pore.
