Molecular Techniques Flashcards
What is a specific endonuclease?
Restriction enzymes produced by bacteria
What do endonucleases do?
Recognise and degradation of foreign DNA
recognise and cut specific DNA
What is special about an endonuclease?
Mainly palindromes
What are restriction enzymes?
Molecular scissors
What is DNA gel electrophoresis?
DNA is separated on the basis of size or shape
What does DNA gel electrophoresis need to work?
Gel- a matrix that allows separation of DNA fragments
Buffer- allows charge on the DNA samples across the gel
Power supply- Generates charge difference across the gel
Stain/detection- To identify the presence of the separated DNA
Why is restriction analysis used?
Investigate size of DNA, mutations, DNA variations, clone DNA
What is a plasmid?
Small circular dsDNA
In bacteria
Can transfer to other bacteria
can have antibiotic resistance
What are the basic steps of gene cloning?
Isolate gene with digestion enzymes
Insert gene into plasmid to form recombinant DNA
Introduce recombinant DNA molecule into suitable cells
identify and isolate the clone contain DNA of interest
Why clone human genes?
To make useful proteins e.g. insulin
to find out what genes do, genetic screening, gene therapy
How is proinsulin produced?
Proinsulin mRNA is reverse transcriptase into proinsulin cDNA
This then joins to a plasmid to make a recombinant plasmid
Then place into bacterium
What are the steps of PCR
- Heat to 95 degrees
- Denature DNA by breaking hydrogen bonds
- Heat to 60 degrees so primers can form hydrogen bonds and anneal with complementary sequences
- Heat to 72 degrees so Taq polymerase adds nucleotides to the 3’ end
- repeated so there will be two double stranded copies of the target DNA
- Half of fragments will have flagging DNA as well as target DNA
Why use PCR?
Amplify specific DNA fragments
investigate single base mutations
investigate small deletions or insertions
investigate genetic relationships
what is protein gel electrophoresis?
Proteins can be separated based on size, shape or charge
What is SDS-PAGE?
separation of proteins based on size
What is IEF?
Seperation of proteins based on charge
What is 2D-PAGE?
Allows separation of complex mixtures of protein
How can you identify a protein?
- Digest a protein with trypsin
- Perform mass spectrometry
- Generate list of peptide sizes
- Use database of predicted peptide sizes for known proteins
- Identify proteins
What is Proteomics?
analysis of all proteins expressed from genome
What is molecular diagnosis?
analysis of a single purified protein
Why are antibodies important?
Bind to specific proteins (antigens) and recognise a few amino acids on a protein (epitope)
What is a polyclonal antibody?
produced by many B lymphocytes, multiple different antibodies, specific to 1 antigen, multiple epitopes
What is a monoclonal antibody?
produced from 1 B lymphocyte, 1 identical antibody, specific to 1 antigen, 1 epitope
What is a western blotting?
Uses antibodies to detect proteins
What is an enzyme assay?
Add samples of enzyme to substrate and measure rate of reaction - can determine how active an enzyme is
How can you detect mutations at a nucleotide level?
DNA sequencing
PCR
Restriction analysis
How can you analyse DNA at a gene level?
Southern or Northern hybridisation
RT-PCR
Microarray
DNA fingerprinting and DNA profiling
How can you analyse DNA at a chromosome level?
Karyotyping
FISH- chromosome painting
How is DNA sequencing done?
Use tagged Dedioxy bases (have a H instead of an OH so chain cannot continue)
These bases are marked and electrophoresis is done to determine DNA order
Important for Genome sequencing https://www.youtube.com/watch?v=SRWvn1mUNMA
What is allele specific PCR?
Use allele specific primers e.g. if we know a mutation in a gene causes a T, would use an A and if this would bind we know have the mutation
What is southern hybridisation?
Used to determine if a specific nucleotide sequence is present
Done by using specific restriction enzymes
electrophoresis is done and transferred to a nylon filter
Radioactive nucleic acid probe is added
Probe binds to complementary DNA sequence
What is northern hybridisation
Same as southern hybridisation but with mRNA
Less stable
What is reverse transcriptase PCR?
Begin with mRNA rather than DNA
What is microarray?
Use when you want to look at 1000s of genes at once
can have whole human genome on a chip
Used to compare two conditions- infected or uninfected
What is DNA fingerprinting?
DNA is cut using a restriction enzyme
Fragments are then separated on the basis of size
Nylon filter is then used
DNA strands are denatured
Probes are added that are complementary to repeat sequences
Xray film
Where probes have bound- appear as dark bands
related- similar banding
What is karyotyping?
Looks at the number and visual appearance of chromosomes
Chromosomes are paired up
can see banding patterns
What is FISH?
Fluorescent in situ hybridisation
Chromosomes are painted so can easily see translocations and tumours
