Module 4 Flashcards
Why does the blue plasmid indicate the presence of transformed bacteria?
Because if LacZ if present, it will encode beta‐galactosidase where it will cleave X-gal to a chromogenic substrate and turn blue. IPTG in media will induce expression of beta‐
galactosidase from lacZ gene
Why does white colonies mean positive clone
Because with LacZ gene disrupted, Functional beta‐galactosidase will not be
expressed and X-gal will not be cleaved. Thus bacterial colonies will be white
What method can be used to differentiate between the DNA
fragments ligated into the cloning plasmid?
RESTRICTION MAPPING
identify position of restriction sites in DNA molecule
determine an insert’s orientation in a cloning plasmid
Selection: identify recombinant plasmids from non‐recombinant plasmids
What are the techniques involved in restriction mapping
i. Digest DNA molecule with REs
ii. Separate digestion products (e.g 3-kbp by digesting with EcoRI)
iii. Size digestion products (Pstl within two EcoRI site)
iv. Infer positions of restriction sites distant between Pstl and each EcoRI)
v. Construct a restriction map
What is REVERSE TRANSCRIPTASE‒PCR (RT‒PCR)
• Reverse transcription of RNA strand into its dsDNA complement
(complementary DNA or cDNA) using reverse transcriptase (RT)
• cDNA is amplified using PCR
What is cDNA
- a DNA copy of mature mRNA
* stable
What is Reverse Transcriptase
- enzyme from retroviruses
* transcribes RNA into DNA
What is the use of RT‒PCR
• diagnosing genetic diseases
• measuring gene expression (can determinate abundance of specific different RNA molecules within a cell or tissue)
• inserting eukaryotic genes into prokaryotes
• studying genomes of viruses whose genomes are composed of RNA,
e.g. Influenza virus A and retroviruses: HIV
What is the process of RT‒PCR
Oligo‐dT primer will bind to the poly-A tail of mRNA and synthesis begins when Reverse transcriptase
+ dNTPs is added.
RNAse H will then digest mRNA purify DNA by column chromatography.
Ligate into linearised plasmid vector
Amplification of cDNA by pCR (Taq DNA polymerase
+ dNTPs)
What is REAL TIME/QUANTITATIVE PCR (QPCR)
Allows simultaneous amplification and quantification
What are the advantages of qPCR over traditional PCR
• Amplification and detection in an integrated system
• Fluorescent dyes/probes allowing constant reaction
monitoring
• Low contamination risk due to sealed reactions
• Rapid cycling times (20‒40 min for 35 cycles)
• High sample throughput (200 to 5000 samples/day)
• Increased sensitivity and reproducibility
• Software driven operation
What are the applications of qPCR
Diagnostic
• Nucleic acid changes (genetic abnormalities) for infectious diseases, cancer
• New strains of viruses (e.g. Influenza)
• Food safety
• Spoilage of foods, levels of fermentation
• Public health protection, e.g. water quality
• Basic research
• Quantitative measurement of gene transcription
• Changes in gene expression over time or in response to a
pharmacological agents
• Progression of cell differentiation in response to changes in
environment
What are the 2 ways to assay
i. Dye assay chemistry using the SYBR Green I dye
Double‐stranded DNA binding dye:
detects accumulation of PCR product during PCR cycles (Will only
fluoresce when
bound to dsDNA)
ii. Probe‐based chemistry using the Taqman probe (Taqman assay)
Fluorogenic probe: enables detection of accumulation of specific PCR product during PCR cycles
What is the basis of the two assays
PCR is the basis of these techniques:
• ssDNA into dsDNA through extension of complementary primers
• Uses DNA polymerase
Fluorescence is the basis of detection
What are the steps for QPCR ASSAY 1: SYBR GREEN I ASSAY
1) Reaction setup: SYBR Green I Dye fluoresces when bound to dsDNA
2) Denaturation: When DNA is denatured, SYBR Green I Dye is released Fluorescence declines drastically
3) Polymerization: During 5’>3’
extension, primers anneal and PCR product is generated
4) Polymerization completed: When polymerization is complete, SYBR Green I Dye binds to dsDNA product – fluorescence increases (measured by computer software)
How do you measure Sybr Green Assay
Intensity of fluorescence
emitted during qPCR
correlates to amount of
DNA product formed
How does TAQMAN ASSAY work
• Exploits the 5‘ nuclease activity of Taq DNA polymerase: cleavage of
oligonucleotide probe during PCR
• Probes:
• are fluorescently labelled at 5’ end
• are chemically modified at their 3’ end
• have a quenching molecule at 3’ end
no fluorescence when probe is in tact (FRET)
• Cleavage of probe generates a detectable signal
What are the steps for TAQMAN ASSAY
1) Polymerization: Fluorescent reporter dye (R) and quencher (Q) are attached to 5’ and 3’ ends of TaqMan probe, respectively. Probe anneals downstream from target sequence. (R) emission is quenched when probe is intact.
2) Strand displacement: 5’ 3’ extension from primer Inclusion of probe does not inhibit PCR
3) Cleavage: During each extension cycle, the DNA polymerase cleaves (R) from
the probe
4) Polymerization completed: Once separated from (Q), (R) emits its characteristic fluorescence
Constructing a cDNA LIBRARY
mRNA template is annealed to a synthetic oligonucleotide (oligo dT) primer
Reverse transcriptase and dNTPs yield a complementary DNA strand.
mRNA is degraded with alkali or RNaseH
To prime synthesis of a second strand, an oligonucleotide of known sequence is often ligated to the 3’ end of the DNA
DNA polymerase I and dNTP extend the primer to yield double-stranded DNA
Plating out a LIBRARY
Take the interested DNA and apply on a new petri dish
Screening DNA Libraries by hybridization
Overlay a nitrocellulose dick to amke a replica of the plate
Remove disk from plate and lyse cells on it and denature DNA with NaOH
Add labelled probe
Colonies with completmentary DNA sequence hybridize to probe
Wash disk, expose to X-ray film
