Session 12+14 (Molecular Techniques) Flashcards
What is needed for protein gel electrophoresis? (4)
Gel, buffer (maintains charge on sample), power, stain
What type of electrophoresis uses proteins intrinsic properties?
Serum protein electrophoresis
density of band = height of graph
What type of electrophoresis separates proteins according to size? How?
SDS-PAGE
Secondary and tertiary structures removed by beta-ME and SDS adds a negative charge to strand
Simply looking at polypeptides
What type of electrophoresis separates proteins according to charge? How?
IEF (isoelectrical focusing)
Gradient of pH is set up (low pH/+charge. High pH/-charge), protein moves until they reach a pH=pI then movement stops because they have no net charge
What type of protein gel electrophoresis separates proteins according to size and charge?
2D-PAGE
Charge (horizontal movement)
Size (vertical movement)
When would you use 2D PAGE?
When you have complex mixtures (ie whole cells)
What is the process of protein identification called?
Protenomics
What is an epitope?
A few amino acids on a protein that antibodies bind to
What are the two main types of antibodies?
Polyclonal - binds to many epitopes
Monoclonal- 1 epitope
When would you use antibodies as probes?
Western blotting
What does ELISA mean?
Enzyme linked immunoabsorbant assay
What are the 3 stages of PCR?
Denaturing
Annealing
Extending
What are the temperatures for the different stages of PCR?
Denaturing- 95
Annealing - 60
Plolymerising - 72
What builds the new strands on DNA in PCR?
DNA Taq polymerase
What sort of primers are needed for PCR?
Forward and reverse
What are the uses of PCR?
Amplify fragment
Investigate single base mutations
Investigate small deletion/insertions
Investigate Variation in genetic relationships
What enzymes cut DNA at specific palindromic sequences?
Restriction endonucleases
Why are DNA fragments negatively charged?
Phosphate group on nucleotides (PO4)
What is the positive electrode called?
Anode
What do you need for DNA gel electrophoresis? (4)
Gel
Buffer
Power supply
Stain
What are the uses of DNA gel electrophoresis? (3)
Investigate size of DNA fragments
Investigate mutations
Investigate DNA variation
What is a plasmid + isolated gene to be copied called?
Recombinant plasmid
What is the vector for cloning DNA usually?
A plasmid
What is used to reseal plasmids?
Ligase enzymes
Why clone human genes? (4)
Make useful proteins
Find out what genes do
Genetic screening
Gene therapy
What do you use to get DNA from mRNA?
Reverse transcriptase
What gene is isolated when making human insulin?
Proinsulin (immature form of the gene)
Why can gel electrophoresis be used to separate molecules of different size?
Large DNA fragments move slowly, smaller fragments move faster
What do restriction enzymes cut?
Phosphodiester bonds
What is DNA sequencing?
Process of determining the precise order of nucleotides within a DNA molecule
How is DNA sequencing done?
Heat DNA + primer + dideoxynucelotides (tagged)
Chains grow
Separate out new strands by gel electrophoresis
Use computers and laser to read
Why are dideoxynucleotides used?
They lack an OH group meaning elongation is terminated
In PCR, at what end does the primer attach and why?
The 3’ end because the DNA chain grows in a 5’-3’ direction
What are allele specific primers?
Sequences that allow for a change in sequence and will bind accordingly
What is southern blotting?
Separating DNA by gel electrophoresis
Transfer to nylon membrane
(Done by soaking gel in alkaline solution- creates ssDNA)
Mix with DNA probes (dont need 100% complementary)
Detection methods detect binding
Why use southern blotting?
Allows for detection of difficult to find DNA
Can be used in conjunction with PCR to detect:
Gene structure expansion and repeats, mutations, genetic variation
How is northern blotting different to southern blotting?
It uses RNA instead of DNA
Why is northern blotting more difficult than southern blotting?
RNA degrades
What sort of primer would you use for RT-PCR? Why?
One with many TTT’s @ 5’ end so it can bind to the polyA tail at the 3’ end of mature RNA
What do you compare during microarray?
2 ‘conditions’
How many DNA sequences do you look at at once in microarray?
Thousands
What does microarray allow us to see?
Gene expression in an individual
What is microarray?
Putting all genes on plate, Take someone's mRNA (from tissue) Use RT to produce cDNA Label cDNA Wash cDNA over plate cDNA will bind to genes Therefore gene expression can be seen
What are mini-satellites?
Repeating areas of base pairs
(Can have the same repeat multiple times at a single loci)
(Variable between homologous chromosome pairs)
How does DNA fingerprinting work?
Use restriction enzymes to chop up DNA
Complete southern blotting
Compare runs
What section of DNA are you looking at in DNA fingerprinting?
The non coding regions of the DNA
What are the probes used for DNA fingerprinting?
Minisatellites
What is karyotyping?
Lining up of chromosomes
What does FISH stand for?
Fluorescent in situ hybridisation
How do you do a FISH?
Make specific DNA sequence copy,
Label probe
Denature DNA and allow hybridisation
Section of DNA becomes visible
