2.7 Molecular bio techniques for clinical microbiology

Question 1

Name 3 molecular biology techniques used in clinical microbiology

Answer 1

PCR, Real time PCR and Microarrays

Question 2

What are the advantages of molecular biological techniques

Answer 2

Speed in provding result
Commercial kits available
Greater sensitivity than conventional methods
Potential for automation

Question 3

What is PCR and what does it enable

Answer 3

Polymerase chain reaction - it enables the amplification of DNA fragments through repeated rounds of DNA synthesis

Question 4

Name the 3 stages of PCR

Answer 4

Denaturation
Annealing
Extension

Question 5

Describe the denaturation stage of PCR

Answer 5

The mixture of target DNA, heat stable DNA polymerase, dNTPs and primers are heated to 95C breaking the hydrogen bonds between bases of the dsDNA and seperating the dsDNA into ssDNA

Question 6

Describe the annealing stage of PCR

Answer 6

The mixture is cooled to 45-60C and primers which are present in excess bind via complementary base pairing to their recognition sites on the ssDNA. The recognition sites must be located at the 3’ ends that flank the target DNA sequence.

Question 7

Describe the extension stage of PCR

Answer 7

The mixture is heated to 72C, at this temperature the taq polymerase uses the primer as its starting point for DNA synthesis. It catalyses the formation of phosphodiester bonds between the primer 3OH and the 5’ phosphate group of a dNTP that is complementary to the corresponding nucleotide of the target DNA to which the primer is bound.

Question 8

How are the products of PCR visualised

Answer 8

it can be visualised by separating it from
unbound primers by gel electrophoresis on the basis of size- the small primers migrate to the end of the gel and the product can be sized by comparison to DNA size markers

Question 9

What is real time PCR

Answer 9

Based on conventional PCR, instead of analysing the final DNA yield with agarose gel electrophoresis. The DNA is quantified during the PCR reaction using fluouresence. The flourescence is acheived by either fluorescent dye which intercalates between bases or dye + labelled DNA probe which are oligonucleotide linked dyes. The flouresence intensity is measured after each cycle.

Question 10

What are the advantages of PCR

Answer 10

As long as the nucleic acid sequence of the target genome is known, primers can be designed to amplify sections of it.
Sample preparation is automated
It provides results very quickly
It can be used to give a quantitative result (real time PCR)

Question 11

What are the disadvantages of PCR

Answer 11

Cross contamination of samples from pipettes etc can give false positives unless strict decontamination procedures are employed.

Impurities in sample can interfere with PCR, sometimes purification of the clinical sample is needed

Question 12

What are the features of a microarray

Answer 12

It is a biochip containing thousands of highly ordered spots arranged into rows and columns. Each spot contains multiple copies of ssDNA called probe DNA, that could for example represent a gene or a portion of a gene of a known pathogen. The location and composition of each spot is recorded in a database.

Question 13

Describe the steps involved in a microarray

Answer 13

1. The target sequences must first be amplified by
   PCR, during which the sample DNA is also tagged
   with a fluorescent marker
2. The amplified DNA is then denatured and allowed
   to hybridise with the DNA on the Chip – unbound
   DNA is washed away
3. The chip is then scanned to see which DNA
   sequences hybridised with the Chip