In-class Test Practice November 2022

Total marks 75

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1. W
a) Using the Beer-Lambert law in your explanation, explain which one of the following
analytes can be most sensitively detected by spectroscopy

Absorption Wavelength Molar absorptivity ()

Analyte
nm L.mol-1.cm-1
Nicotine adenine
340 6220
dinucleotide NADH

Vitamin B2 447 13222

Copper sulfate 660 20

The Beer-Lambert law A = .c.l

Where A = absorbance,  = molar extinction coefficient, c = concentration
of analyte, l = path length of light through the sample

Sensitivity is determined by the value of the molar absorptivity; the higher

the value, the more sensitive the detection.

Molar absorptivity is highest for Vitamin B2; technique most sensitive for this
analyte

4 marks

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2.
a) State the technique that produces the analysis shown below and describe the profiles
shown, for normal samples e.g. in lanes 16 and 17.

Protein electrophoresis using cellulose acetate or agarose. Proteins are stained non-
specifically with amido black B or coomassie blue.
Samples are patient sera.
The profile shows albumin as a thick band at the top followed by 1 (faint band after
albumin), 2 (strong band) ,  (strong band) and  globulins.

6 marks

b) The lowest band in most samples is broad and diffuse. What is present in the band?

The lowest, diffuse band is due to IgG

1 mark

c) Explain why the band is diffuse in most samples.

We normally carry in our blood thousands of different types of IgG each with a
different antigen specificity – polyclonal IgG.

3 marks
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3. Prenatal diagnostics of genetic abnormalities involves a screening programme for
pregnant women.
a) Describe the combined screening test for Down’s syndrome.

Combined test is ultrasound and a blood test for PAPP-A, -hCG

Ultrasound scan (sometimes called a nuchal translucency test) measures the small
collection of fluid at the back of a baby’s neck, which is increased in Downs
Syndrome.

PAPP-A (pregnancy associated plasma protein-A) - low levels associated with

trisomies

free B-hCG (B-human chorionic gonadotrophin). – higher levels in trisomy 21

Combined test picks up 85 to 90% of babies with Down’s syndrome (sensitivity)

6 marks

b) If the test indicates that the fetus may have Down’s syndrome, a genetic test is
offered. Describe one sampling technique to obtain fetal cells for genetic analysis.

CVS - biopsy of placental chorionic villi containing fetal cells exclusively taken at 9.5
to 12.5 weeks gestation; biopsy needle guided through a catheter by ultrasound

OR

Amniocentesis - needle passed into amniotic cavity under guidance with ultrasound
scan. Taken 15th-20th week gestation; samples fetal cells and metabolites from
amniotic fluid

OR

Percutaneous umbilical cord blood sampling - Used if results from CVS or

amniocentesis inconclusive; foetal blood withdrawn from umbilical vein, but difficult to
get many fetal cells

4 marks

c) Explain the steps involved in using fetal cells that would be used for genetic analysis
to confirm Down’s syndrome.

Cells would need to be cultured, then stimulated to grow with a mitogen (PHA
phytohemagglutinin); the cultures are then synchronised to metaphase using
colchicine; lysed by hypotonic swelling, fixed and analysed by FISH

6 marks

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 d) Increasing use of non-invasive pre-natal testing (NIPT) techniques for genetic
screening are being introduced in the NHS to screen for Down’s syndrome. Describe
what is analysed, including an explanation of the origin of the analyte.

Cell free DNA circulating in the blood is analysed; in pregnant women a proportion of
circulating cell free DNA will derive from fetal cells; using qPCR it is possible to measure
the presence of a third copy of chromosome 21 in foetal DNA as indicated by a small
increase the proportion of chrom 21 DNA in the total cfDNA.

marks

e) Comment on the challenges of making NIPT more broadly available for pre-natal
screening of all genetic diseases.

The challenge is to distinguish maternal DNA, which will be the major portion of the
circulating DNA, from fetal DNA; this could be aided by having samples of pure
maternal and paternal DNA for comparison in genotyping. Third generation
sequencing, in which individual strands of nucleic acid are sequenced, may also
help.

4 marks

4. The image below shows the identification of two the loci of two genes in a
chromosomal analysis of a lymphocyte

a) Given that you already have a metaphase spread prepared, describe the key steps of
the technique used to identify the two genes.

The technique is fluorescence in situ hybridisation (FISH).

(Cells cultured, treated with colchicine, hypotonic swelling, lysed, fixed; - metaphase
spread prepared) DNA and probe denatured by treatment with NaOH.
Karyotype would be assessed by chromosome painting – probes of long lengths of
DNA (100-200kb) are prepared from bacterial artificial chromosomes, labelled with a
fluorophore. Different coloured fluorophores are available for each type of
chromosome.

Because chromosomes can be identified along the complete length it is possible to

identify translocations easily, as well as count copies of individual chromosomes.

8 marks

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 b) The white arrows indicate an abnormal pattern. Explain why the pattern is abnormal
and the pathological consequences of the abnormality.

The pink probe detects the gene ABL1 on chromosome 9 and the aquamarine probe
detects the gene BCR on chromosome 22. In the normal chromosomes the two
probes are well separated, but in the abnormal chromosome the two probes are next
to each other. This indicates a translocation between chromosomes 9 and 22, which
leads to chronic myelogenous leukaemia, sometimes called the Philadelphia
chromosome.

5 marks

5. ELISA
a) Describe the steps involved in the sandwich variant of the ELISA technique.
Sandwich ELISA requires two antibodies (Abs) that recognise separate epitopes of the
target antigen.
96-well plate coated with first Ab.
Excess binding sites blocked with protein (albumin or skimmed milk).
Samples and standards containing target antigen are applied.
After incubation excess washed off.
After washing the second antibody is applied – it can have a reporter enzyme attached
(HRP or alkaline phosphatase) attached, or a fluorophore, or a secondary Ab carrying
a reporter enzyme or group can be added.

Excess second Ab washed off

Enzyme substrate added to generate colourimetric signal that can be detected in a
plate reader

10 marks
b) Describe how ELISA can be quantitative.
In order to quantify the amount of a specific protein in a sample it is necessary to use
purified samples of the target protein of known concentration, and to dilute them to
produce a standard curve, usually over a 100-fold range of concentrations.
2 marks

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6. Real-time PCR can be used to detect infection by bacteria.
a) Describe the principles of detection of DNA by real-time PCR.
One format of real-time PCR involves the use of a third primer that binds in the target
amplicon between the forward and reverse primers.
The third primer has a fluorophore at one and a quencher at the other end. When the
third oligo is not bound the fluorescence of the fluorophore is quenched.
During each PCR cycle the third oligo binds and is broken down into individual bases
by the exonuclease activity of the DNA polymerase; the quencher no longer works and
the fluorescent signal of the fluorophore is released.
AS each cycle is completed the fluorescence signal increases.
The relationship between cycle number and fluorescence is sigmoidal, but is
quantitative in the linear portion of the curve.

9 marks

b) How can real-time PCR be used to address the challenge of antibiotic resistance?
In addition to its use to quantify a sample of nucleic acid, real-time PCR can be used
qualitatively too. You need to be aware that antibiotic resistance is often due to
known point mutations in genes that are normally inhibited by the antibiotic.
The PCR detects the presence of a mutation that confers resistance to the antibiotic.

In this format forward and revers primers amplify across the target locus on the DNA.
The oligonucleotide that produces the fluorescent signal can detect a point mutation
that confers resistance.

The technique is shown in slides from lecture 5:

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 3 marks

End of Test

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