Cell Biological Techniques 2 Flashcards
1
Q
What is genomics?
A
The study of an organism’s or a virus’ genome
2
Q
What does genomics involve?
A
- sequencing genomes by determining order of A, G, C, T (U)
3
Q
What is the Sanger method?
A
- Denature template and separate strands
- Build up sequence from primer, copying the template
- ddNTPs to stop strand synthesis
- Read point at which ddNTPs stop reaction
4
Q
What is NGS & what is it used for?
A
- massively parallel sequencing - generate millions of short (50-200bp) sequence reads
- aligned by computers = produce whole genome sequences
5
Q
2 ways genomics is used in cell biology?
A
- mapping of genes linked to inherited disease
2. mapping of somatic DNA changes linked to disease: cancer
6
Q
What do you achieve from examining the cancer cell genome?
A
- information about malignant transformation of cancer cells
- determine how cells have become transformed
- how they have evolved from original cancerous cells
- why they metastasize
- develop new cancer treatments
7
Q
Some info about Hepatocellular carcinoma
A
- changes in exons from HCC were mainly missense mutations (74%)
- insertion/deletion (14%)
- nonsense and splice site mods (12%)
- Wnt/B-catenin pathway affected (becomes disregulated)
8
Q
What are metagenomics?
A
environmental, community or population genomics
- analyses a collection of genetic material in a sample
- first developed to examine non-culturable organisms
9
Q
What are transcriptomics?
A
- Study of mRNA molecules inside organisms, tissues and cells
- exceptions, but GENOME should be same in all cells
- TRANSCRIPTOME - different in cells - represents point in time of gene expression levels, will change over time
10
Q
How do you analyse the transcriptome?
A
- DNA microarray
- RNA sequencing (more common)
- info about which genes are expressed & relative levels
11
Q
What are cDNA microarrays?
A
- convert mRNA from 2 samples to cDNA
- cDNA is labelled with different fluorescent dye
- labelled cDNA spotted on glass slide already spotted with 1000s of specific cDNA probes
- ratio of red & green fluorescence intensities - indicative of relative abundance of corresponding DNA probe in 2 samples
12
Q
What is RNA-Seq?
A
- analyses cDNA copied from RNA by NGS
- sequence reads mapped onto reference genome
- info about genes expressed
- info about RNA splicing
- No. sequence reads = amount of RNA
13
Q
What are proteomics?
A
- study of large no. of proteins in whole organisms, tissues, cells
- How the levels of these change in response to external factors
- proteome will differ - rflect specialist cell function & diseases state
- can identify up to 1000s of proteins in a sample
14
Q
What technique is central to proteomic studies?
A
Mass spectrometry of trypsin digested proteins
15
Q
How does mass spectrometry identify proteins?
A
- proteins digested by protease
- SOMETIMES - masses of peptides alone can identify protein
- peptide mass fingerprint used to search in database for protein
