Molecular Diagnosis

Question 1

What can we use to analyse proteins?

Answer 1

protein electrophoresis, immunoassays and enzyme assays

Question 2

Why do bacteria naturally produce endonucleases?

Answer 2

It is the normal mechanism for recognising and degrading foreign DNA in bacterial cells.

Question 3

How do bacteria protect their own DNA from endonucleases?

Answer 3

Methylation

Question 4

What is another name for the positive electrode?

Answer 4

Anode

Question 5

What are the requirements for DNA gel electrophoresis?

Answer 5

gel: matrix for fragment separation
buffer: allows DNA to maintain charge
power supply: generates charge difference across gel
stain

Question 6

How do we manufacture insulin?

Answer 6

take mRNA for proinsulin from pancreas
use reverse transcriptase
create cDNA of proinsulin
join to a plasmid (recombinant DNA)
inject into bacterium 
production of proinsulin occurs

Question 7

What is the sequence of events for PCR?

Answer 7

heat to 95’C: break H bonds
lower to 55’C: allow DNA primers to anneal
heat to 72’C: optimum temp for Taq DNA polymerase

Question 8

How many primers do we need for PCR?

Answer 8

2

They define the region we want to amplify.

Question 9

Which direction does polymerase work?

Answer 9

5’ -> 3’

Question 10

What is different with protein electrophoresis to DNA?

Answer 10

gel is upright with wells at the top

electrodes are placed dependent upon intrinsic protein charge

Question 11

What is the major protein constituent of serum?

Answer 11

Albumin

Question 12

How does SDS-PAGE work?

Answer 12

proteins separated purely on the basis of size
denature proteins so that they have a uniform negative charge
movement towards positive electrode based just on size

Question 13

What is isoelectric focusing and how does it work? (IEF)

Answer 13

proteins separated on the basis of charge
stable pH gradient established
proteins migrate until they reach pH = to their pI
at their isoelectric point they have no net charge so stop migrating

Question 14

What is 2D PAGE and how does it work?

Answer 14

2 step separation of proteins
use isoelectric focusing first to separate by charge
followed by SDS-PAGE to separate by size
allows separation of complex mixtures

Question 15

Define proteomics

Answer 15

Analysis of all proteins expressed from a genome

Question 16

How do we perform proteomics?

Answer 16

digest protein with trypsin
perform mass spectrometry
generate list of peptide sizes
use database to identify proteins

Question 17

What are the features of polyclonal antibodies?

Answer 17

produced by many B lymphocytes
multiple different antibodies
specific to 1 antigen each
have multiple epitopes

Question 18

What are the features of monoclonal antibodies?

Answer 18

produced from 1 B lymphocyte
1 identical antibody
specific to 1 antigen and 1 epitope

Question 19

What do we detect with Western Blotting and how does it work?

Answer 19

proteins detected, proteins separated by SDS-PAGE, bound to more solid matrix, incubated with primary antibody for protein interested in, add enzyme linked secondary antibody that binds to primary that contains a marker

Question 20

What is ELISA and how does it work?

Answer 20

Enzyme-linked immunoabsorbant assay
antigen coated wells
specific antibody binds to antigen
enzyme-linked antibody binds to specific antibody
substrate is added and converted to a coloured product by the enzyme
rate of colour formation is proportional to amount of specific antibody

Question 21

How do radioimmunoassays work?

Answer 21

same way as ELISA

uses radiolabelled primary antibodies

Question 22

What would you look for after an MI and using which technique?

Answer 22

creatine kinase and cardiac troponin 1

ELISA

Question 23

What techniques can we use to analyse DNA at a chromosome level?

Answer 23

karyotyping and FISH

Question 24

Why is a mismatch of a probe at the 3’ end more dramatic?

Answer 24

DNA polymerase cannot bind and extend so there will be no PCR product

Question 25

What do we use Northern hybridisation for?

Answer 25

analysis of RNA

Question 26

What is RT-PCR?

Answer 26

reverse transcriptase PCR
just an added step to the normal PCR
use mRNA and enzyme to make cDNA before PCR

Question 27

Why are primers for mRNA easy to make?

Answer 27

all mRNAs have a polyA tail

therefore we can make a polyT primer

Question 28

Why would we use microarrays?

Answer 28

investigate thousands of genes simultaneously
comparison of 2 conditions (disease/normal or disease/disease)
investigating control of gene expression

Question 29

How does DNA fingerprinting work?

Answer 29

there are lots of repeated sequences in the human genome
these are called mini-satellites
mini-satellites show copy number variations
it shows family relationships

Question 30

How is DNA profiling different from DNA fingerprinting?

Answer 30

DNA profiling uses smaller repeat sequences

these smaller sequences are called STRs = small tandem repeats

Question 31

What do we use karyotyping for?

Answer 31

numbering and pairing chromosomes

looking at the appearance of chromosomes

Question 32

What are the advantages of FISH?

Answer 32

we can look at chromosomes without destroying them

fluorescent dyes allow us to see translocation

Question 33

What can we use to analyse DNA at a gene level?

Answer 33

restriction enzymes
DNA gel electrophoresis 
PCR 
Southern hybridisation 
DNA fingerprinting/profiling 
microarray