Genomics, transcriptomics, proteomics and PCR Flashcards
What is Genome transcribed?
Transcriptome - RNA copies of the active protein-coding genes
What is Transcriptome translated ?
Proteome - The cell’s repertoire of proteins
Name steps of Whole Genomic Sequencing (WGS)?
- Genomic DNA
- Tagmentation of Genomic DNA
- Sequencing
- Data Analysis & Report
RNA sequencing in in vivo ?
- Transcription
- Pre-mRNA
- Intron splicing
- Mature mRNA
RNA sequencing in in vitro ?
- RNA fragments
- Reverse transcription
- ds-cDNA fragments
- High-throughout sequencing
RNA sequencing in in silico ?
- Alignment
- Splice variants
RNA sequencing can not only identify which transcripts are in each sample but can ?
Also quantify amounts
- This allows us to compare expression levels between different samples
RNA sequencing can not only identify which transcripts are in each sample but can ?
Also quantify amounts
- This allows us to compare expression levels between different samples
Proteome methods ?
- Antibody arrays
- 2D gel electrophoresis
- 2D DIGE
Stages of PCR reaction ?
a) melting
b) annealing
c) extension
What do the end of the amplified DNA molecules represent ?
The primers not the original DNA sequence
A thermostable DNA polymerase is used that ?
thermostable DNA polymerase is used that is not denatured at 94ºC allowing automation of the cycling process. Different polymerases are available depending on the required length and accuracy of the amplified DNA fragment
What is Taq ?
Taq is the original enzyme but is highly error prone, others like Pfu are less productivebut have better proof-reading capabilities. Mixtures are commonly used
Identification / characterisation of DNA sequences ?
A. Confirming presence of sequence (use of nested primers)
B. Characterising length polymophisms (DNA Fingerprinting)
C. Isolation of polymorphic alleles for further analysis
Cloning of DNA sequences ?
A. ‘Blunt’ ends – use of T4 DNAP or T-vectors
B. Introducing restriction sites in primers
Explain RT-PCR ?
- Anneal primer to mRNA and extend with Reverse Transcriptase. Primer could anneal to polyA tail or could be gene-specific
- Anneal 2nd primer and amplify with Thermostable polymerase. 2nd primer in this case is gene-specific. Note that the Tth thermostable DNA polymerase also has reverse transcriptase activity and so can be used for both RT and PCR steps.
- Alternatively a polynucleotide tail can be added to the 3’ end of the first DNA strand, then the cDNA can be amplified using polyC and polyT primers
- Final PCR product can be cloned, sequenced etc
The amount of PCR product formed in an amplification reaction is not proportional to?
The amount of starting template. This makes it very difficult to use PCR as a means of quantifying starting material, for instance the levels of a particular mRNA in a sample
How can PCR be quantitative? and how can this be achieved?
- If the rate of increase of product is determined
- This can be achieved most efficiently by measuring the production of PCR product with a fluorescent marker. Fluorescence can either me measured after each cycle or at the end of the PCR reaction
There are a number of ways of detecting the PCR product:
1) DNA binding dyes – only fluoresce when bound to double-stranded DNA
2) “Taqman” assay – PCR reaction removes a fluorescent quencher
3) Molecular Beacons – self quenching fluorescent probes fluoresce during annealing
DNA Binding dyes - The LightCycler Assay
SYBR Green is one of the most commonly used fluorescent dyes in qPCR. It binds to double-stranded DNA molecules by intercalating between the DNA bases. Once intercalated to DNA, SYBR Green becomes less mobile, causing its energy to be released as fluorescence
The Taqman Assay ?
TaqMan PCR is a type of real-time PCR and uses a nucleic acid probe complementary to an internal segment of the target DNA. The probe is labeled with two fluorescent moieties. The emission spectrum of one overlaps the excitation spectrum of the other, resulting in “quenching” of the first fluorophore by the second
What are Molecular Beacons?
Molecular beacons are hairpin-shaped molecules with an internally quenched fluorophore whose fluorescence is restored when they bind to a target nucleic acid sequence. This is a novel non-radioactive method for detecting specific sequences of nucleic acids
