Lecture 25-29 Flashcards
What is a SNP
single nucleotide polymorphism. Changes single nucleotide in strand.
What can genetic variation be detected by
Southern blotting, FISH and PCR
What is a hybridisation method
Northern and Southern blotting where the DNA/RNA of interest is single stranded and a complementary radioactively labelled probe hybridises with the DNA/RNA of interest and forms double stranded DNA
What is a bacterial expression vector
A plasmid designed for protein expression in bacterial cells. The vector is used to introduce a specific gene into a target cell, and can make the cell produce the specific protein encoded by the specific gene.
What do southern, northern and western blotting techniques detect?
Southern: DNA fragment
Northern: RNA fragment
Western: Protein
Describe process of western blotting
- Extract cell from tissue or recombinant organism
- Seperate proteins with gel electropherisis
- Transfer protein from gel to filter (using electric current.)
- Use primary antibody as a probe which binds to desired specific proteins
- Radioactive labelled secondary antibody binds to primary antibody/antigen complex
- Detect protein of interest through autoradiography through the radioactive labelled secondary antibody.
Describe process of southern (DNA) and northern (RNA) blotting
- Purify and Digest DNA/RNA with restriction enzymes
- Separate DNA/RNA fragments by gel electrophoresis
- Denature DNA in gel using NaOH
- Wash and neutralize gel using a buffer
- Overlay with nitrocellulose filter
- DNA/RNA transfer from gel to filter
- Hybridise filter with a radiolabelled probe
- Detect specific DNA/RNA bands by autoradiography
What are microsatelites/short tandem repeats (STRs)
STRs are short DNA sequence repeats which can be repeated in same location. The number of STRs differ between individuals, they give genetic variation to individuals. They can be detected by restriction endonuclease digestion followed by southern blotting or PCR (PCR used to analyse crime scene samples).
Advantages of PCR for detecting STRs in crime forensics
- Can amplify DNA from a small sample\
- Results are obtained quickly
- Reaction is highly sensitive, copies only target sequence.
How are you able to detect a restriction fragment length polymorphism (RFLP) by southern blotting?
A RFLP deletes a restriction site in the DNA. This means that after southern blotting, The individual with the RFLP will have one fragment (eg 6 kb) and the individual without the RFLP will have two fragments (2 and 4 kb).
What is a RFLP?
A restriction fragment length polymorphism (RFLP) is a SNP that alters a restriction site resulting in variation of sizes of fragments as a restriction site is missing.
What does a bacterial expression vector require to express the protein? (transcription of the cDNA)
- Promoter to initiate transcription
- Transcription terminator to terminate transcription
- Ribosome binding site required for translation initiation
- Start and stop codon to start and terminate translation
What do you need to insert cDNA into the bacterial expression vector
Multiple cloning site (where the cDNA goes) and the insertion of the cDNA needs to be directional (directional cloning)
What is FISH
FISH is a hybridization method which uses fluorescent labelled DNA probes complementary to desired specific DNA sequences. Used to detect presence or absence of DNA sequences
What is the procedure of FISH
- DNA is denatured to single strand
- Cell is flooded with fluorescent labelled DNA probes (single stranded) with the complementary sequence to desired DNA.
- The probes bind to specific DNA and the rest are washed away.
- Fluorescent molecules are added which binds to fluorescent labelled probe which allow visualization of desired DNA.
