Session 7/8 - incomplete Flashcards
(35 cards)
0
Q
How are proteins analysed?
A
- Protein electrophoresis
- Immunoassays
- Enzyme assays
1
Q
How is DNA analysed at gene levels?
A
- Restriction enzymes
- DNA gel electrophoresis
- PCR
2
Q
How is DNA analysed at nucleotide level?
A
- DNA sequencing
3
Q
How is DNA analysed at gene level?
A
- Southern hybridisation
- Microarray
- PCR variations
4
Q
How is DNA analysed at chromosome level?
A
- Karyotyping
- FISH
5
Q
What are restriction enzymes?
A
- Endonucleases
- Endonucleases are specific
- Recognise and cut specific DNA sequences (restriction sites)
- Act as ‘molecular scissors’
- Staggered cuts - ‘sticky ends’
- Produced by bacteria
6
Q
How can the formation of ‘sticky ends’ by endonucleases be reversed?
A
- DNA ligase enzyme
- Joins cut ends together or a different fragment
- DNA should be cut by the same endonucleases so that the sticky ends will be the same on each fragment (some fragments cut with different endonucleases will still have complementary stick ends)
7
Q
What is restriction analysis?
A
- Using restriction endonucleases, DNA ligase and electrophoresis to:
~ investigate the size of DNASE fragment (eg in deletions)
~ investigate mutations (eg sickle cell disease)
~ investigate DNA variation (eg DNA fingerprinting)
~ gene cloning
8
Q
What is DNA gel electrophoresis and what does it need?
A
- Used to separate different sized DNA fragments
- Requires: gel; buffer; power supply; stain
9
Q
What is the process of DNA gel electrophoresis?
A
- A solution of different fragment is placed in a well at the negative electrode end
- A charge encourages the negatively charged DNA to move through the gel towards the positive electrode
- Larger fragment move slower, so the fragments separate out
- Fragments of known size are used as a reference
10
Q
What is gene cloning?
A
- Uses plasmids from bacteria
- Plasmids are small circular DNA, can be transferred to other bacterial and can contain antibiotic resistant genes
- Antibiotic resistant gene is used to positively select for bacteria that have taken up the plasmid
11
Q
What is the process of gene cloning?
A
- Plasmid is cut using restriction endonucleases
- Gene of interest is added -> becomes a recombinant DNA molecule
- Introduced into bacterium- called transformation
- Select for cells containing recombinant DNA using resistant antibiotic genes
- Bacteria containing recombinant DNA are placed in an environment to multiply
12
Q
Why are human genes cloned?
A
- Make useful proteins eg insulin
- Find out what genes do eg HTT
- Genetic screening eg Huntington’s, cystic fibrosis
- Gene therapy? eg cystic fibrosis
13
Q
What are the functions of the polymerase chain reaction?
A
- Amplify a specific DNA fragment
- Investigate single base mutations eg Tay Sachs, sickle cell disease
- Investigate small deletions or insertions eg cystic fibrosis
- Investigate variation and genetic relationships eg DNA profiling, DNA typing
14
Q
What is required for PCR?
A
- DNA polymerase (thermostable Taq DNA polymerase from thermus aquaticus
- Pair of primers (forward and reverse) to define the region to be copied
- Temperature cycles that allow the DNA to denature, anneal and polymerise
15
Q
What is the process of PCR?
A
- Denaturation at high temperature (94-96oC): hydrogen bonds between complementary bases in DNA break to separate strands
- Renaturation at lower temperatures (50-60oC): Primers anneal to complementary section of DNA by forming hydrogen bonds with the ends of the target sequence
- DNA synthesis at medium temperatures (75-80oC): DNA polymerase adds nucleotides to the 3’ end of each primer
16
Q
What is protein gel electrophoresis?
A
- Proteins can be separate on the basis of size, shape or charge
- Proteins are charged molecules so will move towards the cathode or the anode if placed in an electric field
17
Q
What types of protein gel electrophoresis are there?
A
- SDS-PAGE
- Isoelectric focusing
- 2D-PAGE
18
Q
What is SDS-PAGE?
A
- Technique that allows proteins to be separated purely by molecular weight
19
Q
What is the process of SDS-PAGE?
A
- Detergent SDS (sodium dodecyl sulphate) denatures protein molecule and gives the protein a negative charge proportionate to the molecular weight
- Electrophoresis takes place: proteins migrate from positive to negative electrode and the proteins with the largest molecular weight travel further (as they have a more negative charge from SDs)
20
Q
What is Isoelectric focusing?
A
- Separate proteins by their isoelectric points (pI)
- Proteins are applied to a gel containing a pH gradient
- Proteins will migrate until it reaches a pH that matched its pI (here it has no overall charge and so will stop migrating)
21
Q
What is 2D-PAGE?
A
- Allows the separation of proteins with identical pI values but different weights or vice versa
- Gel is turned 90o and run for a different property to separate bands out
- Important technique for proteomics
22
Q
Why is the measurement of the activity of an enzyme clinically useful?
A
- Indicates whether the enzyme is present at normal levels
- Activity of serum enzymes are often measured as they are indicators of tissue damage
- Indicates tissue damage is caused by a disease of an enzyme normally found in a particular tissue is at elevated levels in the serum
23
Q
How are enzyme assays carried out?
A
- Performed at optimal pH, temperature and ionic strength
- Appropriate ions or cofactors needed for enzyme action must be included
- Production of product/removal of substrate is measured
24
What are some examples of clinically serum enzymes and what they are a marker for?
- Aspartate transaminase (AST)/Alanine transaminase (ALT): liver damage/disease
- Creatine kinase (CK)/Lactate dehydrogenase (LDH): Myocardial infarction
- Amylase/lipase: Pancreatitis
25
What is Western blotting?
- Follows SDS-PAGE
- Separated proteins are transferred to a nitrocellulose membrane
- Specific proteins can be visualised by binding with antibodies conjugated to a label (eg fluorescence)
26
What are Enzyme-Linked Immunoabsorbant assays (ELISA) and how are they carried out?
- Concentration of a protein can be detected by analysing binding of its corresponding antibody
- The first (primary) antibody specific to the protein is immobilised on a solid support (eg microtitre wall)
- Solution to be assayed is applied to the antibody-coated surface
- A second (secondary) antibody conjugated with an enzyme binds to the antibody-antigen complex
- Binding of the second antibody is measured by assaying for the enzymes activity (ie how much product is produced/substrate is removed)
27
How is DNA sequencing carried out with the Sanger Dideoxy Chain Termination Method?
- Fluorescent/radioactively stained ddNTPs are added (with regular dNTPs) to a DNA template strand along with DNA polymerase to create a complementary DNA a strand
- ddNTPs (dideoxynucleoside triphosphate) are a variation of dNTPs that lack a 3' OH group so polymerisation cannot occur and the strand terminates. Depending on which ddNTP is used (ddATP, ddGTP...) the new strand will stop at different places
- This produces lots of new DNA fragment of different lengths (and therefore different sizes) that can be denatured with heat and separated using gel electrophoresis. Each of the different ddNTP runs in a different lane and the end result allows us to write the DNA sequence
28
What is Southern Blotting used to investigate?
- Gene structure eg large deletions/duplications
- Gene expansions or triplet repeats eg fragile X syndrome
- Variation eg DNA fingerprinting
29
How is Southern Blotting/Hybridisation carried out?
- Unlabelled DNA from gel electrophoresis can be marked during southern blotting
- Nylon is used to transfer the fragments from electrophoresis
- The fragments are then hybridised with a labelled gene probe to show the specific DNA fragments
- The use of radioactive probes can mark specific complimentary DNA fragments
30
What is Northern blotting?
- Adding a radioactive probe to mark RNA
31
What is Western blotting?
- Like Southern blotting but with proteins
32
How can PCR be used in an allele-specific test?
- Use primers specific for sequence either side of the allele of interest to amplify the specific allele
33
How can restriction analysis be used in an allele-specific test?
- Use one/multiple restriction enzyme with restriction sites around/within the allele
- Analyse the size of fragments produced
- If the restriction enzyme cuts the wild type but not the sample, the restriction site is mutated/missing
34
How can DNA hybridisation be used in an allele-specific test?
- Use a DNA probe that is complementary to either the wild type allele or mutated allele and see which binds
