L8: Molecular Bio Techniques II Flashcards
What is northern hybridisation?
Allow identification of RNA molecules containing specific sequences
Describe the northern hybridisation blot
mRNA is extracted from tissue/sample & separated by size on an agarose gel
- mRNA is transferred directly to membrane
- a labelled DNA probe from specific gene is hybridised to RNA on membrane
What does in situ hybridisation involve?
Involves hybridising a probe directly to RNA without blotting
Why is in situ hybridisation helpful?
Identify where & when genes are transcribed in embryos in developmental genetics
What labellings are used in ‘in situ hybridisation’
Digoxygenin
OR
Fluorescently labelled probes
What is myogenin required for?
Muscle development
What does DNA sequence provide information about?
How the genome is organised
What can DNA sequencing predict?
Sequence of amino acids contained in the protein that the gene encodes
Describe dideoxynucleotide chain-termination sequencing
Allows rapid sequencing of large amounts of DNA
- makes use of ‘dideoxy’ nucleotides which interrupt the ability of DNA polymerase to copy DNA
How is DNA usually replicated using?
Deoxynucleotides (dNTPs)
How does DNA polymerase incorporate nucleotides by?
FOrmation of phosphodiester bond between 5’ PO4 group of nucleotide being incorporated & 3’ OH on previous nucleotide
Name of the nucleotide that DNA sequencing uses which do not have the 3’ -OH?
Dideoxynucleotides (ddNTPs)
Why is DNA polymerase not able to incorporate further nucleotides after incorporation of a ddNTP?
No 3’OH so cannot form phosphodiester bond
How does dideoxynucleotide sequencing work?
4 parallel sequencing reactions are done (A,C,G,T)
- Each reaction contains many copies of DNA fragments
- Double stranded DNA separated into single strands
- All dNTPSand ddATP/ddCTP are added together
What role do dideoxynucleotides play in DNA sequencing?
Terminate DNA synthesis at specific bases
What type of RNA molecules are identified using Northern hybridization?
Messenger RNA (mRNA)
What is the significance of the Sanger method in DNA sequencing?
. Enabling accurate DNA sequencing
Difference between Sanger sequencing & NGS
NGS reads millions of DNA fragments simultaneously
- Short reads but very fast
Purpose of PCR
Amplify specific DNA fragments** without cloning **
3 stages of PCR
1) Denaturation
2) Annealing
3) Extension
What happens during denaturation in PCR?
95 degrees
- DNA strands separate
What happens during annealing in PCR?
45-68 degrees
- Primers bind to target DNA
What happens during extension in PCR?
72 degrees
Taq pol synthesises new DNA
2 features of PCR
1) Highly sensitive: amplify DNA from tiny samples
2) Exponential amplication: after 30 cycles, produce 10^9 copies
Amplications of PCR
1) Forensics
2) Medical Diagnosis
3) Paternity Testing
4) Ancient DNA studies
Describe the relationship between PCR produce size & amount of DNA between primers
PCR produce size = amount of DNA between primers
What are amplified DNA fragments separated by?
Gel electrophoresis
