Biochemistry Practicals Flashcards
What is glycosuria?
sugar in urine
What is glycosuria indicative of?
diabetes mellitus
What is one of the most common hormonal disorders of cats?
diabetes mellitus
What hormone are diabetic cats deficient in/resistant to?
insulin
What produces insulin?
the pancreas
what is insulin produced in response to?
in response to an increase in blood glucose
What does insulin help to maintain in the body?
normal levels of blood glucose
How does insulin help to maintain normal levels of blood glucose?
allows glucose to be taken up by cells in the body (and used for energy)
What happens in diabetic cats where there is no insulin produced or they are resistant to it?
unable to respond to increases in blood glucose levels and become hyperglycemic
Where can glucose be excreted if hyperglycemia becomes severe?
in the urine
What do reducing sugars have free in their molecular structure?
free aldehyde
In what sort of reaction can an aldehyde be oxidised?
radox
What happens during a radox reaction where an aldehyde is oxidised?
another compound is reduced
What molecules are capable of isomerizing to produce an aldehyde group in solution?
sugars with ketone groups in their open chain form
what colour is Benedict’s reagent?
blue
What does Benedict’s reagent contain?
sodium citrate, sodium carbonate and copper sulphate
what is Benedict’s reagent used to test for?
presence of reducing sugars
What happens if a solution of Benedict’s reagent and a reducing sugar are heated?
copper (II) ions are reduced to copper (I) ions
What effect does the reduction of copper (II) ions to copper (I) ions have on the colour of the solution?
changes from blue to green to orange to red-orange to brick red
What precipitate may appear in the bottom of a test tube when a solution of Benedict’s reagent and a reducing sugar is heated?
copper (I) oxide
What does the amount of precipitate in the bottom of the Benedict’s reagent solution illustrate about the amount of reducing sugar present in the solution?
more precipitate means more reducing sugar
What is the equation for the Benedict’s reagent half reaction with reducing sugars?
2Cu2+ + 2e- = 2Cu+
what is proteinuria?
protein in urine
What are the potential causes of proteinuria?
many but one of the main ones is chronic kidney disease
What happens in normal kidneys to stop proteinuria?
serum proteins are too large to pass across the glomerulus into the tubules to become urine, any that do pass across the glomerulus are usually reabsorbed or broken down
What is a glomerulus?
bundle of capillaries in the kidney
Why can protein end up in the urine if kidneys are damaged?
large proteins may be able to pass into the tubules and there is poor reabsorption of proteins back into the blood and so protein appears in urine
What is the biuret test a chemical test for?
detecting presence of peptide bonds
What happens to copper (II) ion during a positive biuret test?
reduced to copper (I) forms a complex with nitrogens and carbons of the peptide bonds in an alkaline solution
How is the presence of proteins indicated in a biuret test?
violet colour
What can develop in the urinary tracts of cats?
clusters of crystals/stones
What can clusters of crystals or stones in the urinary tract of cats cause?
serious complication particularly if they pass into the ureter and urethra where they may cause a blockage
What are the 2 most common types of crystals/stones found in cats urinary tracts?
struvite, oxalate
When are struvite crystals/stones most likely to develop in the urinary tract?
urine with a pH above 7
When are oxalate crystals/stones most likely to develop in the urinary tract?
urine with a pH below 6
What is Bromothymol a test of?
pH
What sort of indicator is bromothmol?
pH
What colour does bromothmol become with a acidic (decreasing) pH?
yellow
When is colour change in bromothmol difficult to see with the naked eye?
over a narrow range of pH where the colour change is likely to be small
What is spectrophotometry used for?
to quantify the colour of a solution to produce a measurement of light absorption
What can we use absorbances from pH standards to do?
plot a standard curve and use it to work out the pH of samples by their absorbance
Why is DNA difficult to isolate intact and undameged?
it is large and fragile
in what state do isolation procedures provide DNA?
biologically active form but not completely undamaged. The DNA yielded is stable, of high molecular weight and relatively free of RNA and protein
In what sort of solution is DNA more stable?
salt solution
What happens in the first stage of a general method for isolation of DNA from microorganisms?
disruption of cell wall and release of DNA into medium where it is soluble and protected from degradation
What is the medium used in general method for isolation of DNA from microorganisms?
buffered saline solution containing EDTA
What are the 2 functions of EDTA in general method for isolation of DNA from microorganisms?
1) binds divalent metal ions that could form salts with anionic phosphate groups of DNA
2) inhibits deoxyribonucleases that require Mg2+ or Mn2+ (divalent ions)
What divalent metal ions are present in EDTA?
Ca2+, Mg2+ and Mn2+
What pH is the buffered saline solution used in general method for isolation of DNA from microorganisms?
pH 8 (mildly alkaline)
What effect does mildly alkaline pH have on DNA during general method for isolation of DNA from microorganisms?
reduces electrostatic interaction between DNA and basic histones and polycationic amines (spermine and spermidine).
also diminishes nuclease activity and denature other protein
What happens in the second stage of a general method for isolation of DNA from microorganisms?
dissociation of the protein-DNA complexes
What do detergents do during the second phase of the general method for isolation of DNA from microorganisms?
stabilise inner membrane and dismiss ionic interactions between positive histones and negative DNA backbone
What anioinic detergent is used during the second stage of a general method for isolation of DNA from microorganisms?
SDS - sodium dodecyl sulphate (SDS)
What does SDS do during the second stage of a general method for isolation of DNA from microorganisms?
binds to proteins and gives them extensive anionic character (makes them negatively charged)
What is a secondary effect of SDS?
denatures DNA and other proteins
What does an alkaline pH favor in protein-DNA complexes?
dissociation of these complexes
Why is an increased salt concentration added to DNA solution in isolation of DNA from organisms?
to ensure complete dissociation of DNA-protein complex and remove bound cationic (positive) amines
What happens in the third stage of a general method for isolation of DNA from microorganisms?
separation of DNA from from other soluble cellular components
What must happen before precipitation of DNA in the third stage of isolation of DNA from microorganisms?
treatment with chloroform-isoamyl alcohol followed by centrifugation
How many layers are produced from centrifugation of DNA in the third stage of isolation of DNA from micro-organisms?
3 layers
What are the 3 layers produced from centrifugation of DNA in the third stage of isolation of DNA from micro-organisms?
upper aqueous phase
lower organic phase
compact band of denatured proteins
What effect does chloroform-isoamyl alcohol have in the third stage of isolation of DNA from micro-organisms?
chloroform causes surface denaturation of proteins
isoamyl reduces foaming and stabilises interface with phases
How are nucleic acid seperated and DNA precipitated in the third stage of isolation of DNA from micro-organisms?
addition of isopropanol
What is a consequence of DNA’s ionic nature?
becomes insoluble if aqueous medium is made less polar by the addition of organic solvent
What is PCR a method for?
amplifying very small amounts of DNA to levels that can be easily detected in a short period of time
What do PCR primers bind to?
a unique fragment of DNA
What are PCR primers used for?
to amplify DNA found
Can PCR be used in diagnostics?
yes
What are the components involved in PCR?
DNA template
DNA polymerase
Primers
Nucleotides
What is the DNA template?
sample containing the target DNA
What is DNA polymerase?
heat resistant enzyme able to synthesise new strands of DNA complementary to the target sequence using DNA template and primers
What are primers?
pair of short pieces of single stranded DNA, each piece complimentary to one end of the target sequence
What happens when DNA polymerase meets a primer that is base paired with a longer piece of DNA?
it attaches itself near the end of the primer and starts adding nucleotides
What are nucleotides?
single units of the bases A, T, G and C which are building blocks for new DNA strands
What are the 3 stages of PCR?
denaturation, annealing, elongation
What happens during the denaturation phase of PCR?
DNA is heated to approx. 95 degrees C to separate DNA template strands
What happens during the annealing phase of PCR?
DNA is cooled to approx 55 to 68 degrees C to allow the primers to stick to each end of the target DNA
What happens during the elongation phase of PCR?
DNA is heated to approx 72 degrees C. DNA polymerase binds to the end of the primers and starts to synthesise the new strand of DNA. Primers are extended into full length copies of the target region
What stages of PCR are one cycle?
all stages 1-3
How long is a round of PCR expected to take?
1-2 mins to complete
How many PCR cycles are involved in a typical reaction?
35-40 cycles
How does the number of copies during each cycle of PCR increase?
exponentially (doubles each time)
Why are PCR products run on agarose gel?
to determine the presence of specific DNA in the original sample
What will a positive PCR result show on agarose gel?
presence of a band which is the same length as the specific fragment of DNA which was amplified
Are UTIs more common in male or female cats?
obstruction more common in males
What are the common bacterial causes of UTI?
EColi
can also be Staphylococcus spp and Streptococcus spp
What are the clinical signs of a UTI?
straining to urinate blood in urine urinating frequently and in small amounts crying when urinating urinating in inappropriate places licking the genitals discomfort
Why must the presence of bacteria be identified before treatment for UTI can begin?
the clinical signs can occur due to a number of urinary tract disorders not just UTI
