Lecture 8

Question 1

Good laboratory practices for PCR

Answer 1

The procedure of PCR is a very sensitive method.
Utmost care should be taken to prevent any type of carry over or contamination while working.
The results depend on the quality of reagents, equipment ,test procedures, work area cleanliness
As a result all QC and QA parameters should always be followed
Workflow between rooms must be uni -directional from clean areas to contaminated areas.
Extraction of Nucleic acid from the clinical samples must be performed in a clean area where PCR stocks, reagents and other material have not been handled.
The procedure requires the use of very small( minute) quantities, so one should be careful in the use of micropipettes, tips and disposal of the contaminated tips.

Question 2

Step 1 : of PCR

Answer 2

Denaturation
Breaking the double helix structure of DNA
Denaturation happens at a high temperature of 94º C

The vast majority of PCR methods rely onThermal cycling.

Multiplying DNA in vitro consists of three repetitive steps:

Question 3

Step 2 : PCR

Answer 3

Step 2 : Annealing or Priming
Short DNA fragments (Primers) complimentary base pair to the sequence of interest
Annealing occurs at cooler temperatures between 55-65º C

Question 4

Step 3 : PCR

Answer 4

Step 3: Extension
The enzyme Taq polymerase catalyzes the attachment of dinucleotide triphosphates (dNTP), extending the DNA strand
Extension occurs at 72º C

Question 5

Genetic Material DNA vs RNA

Answer 5

DNA – Deoxyribonucleic acid
RNA – Ribonucleic acid
DNA is a double stranded helix
RNA is a single stranded helix
DNA has a set of nucleotides which contain genetic material
RNA principle acts as a messenger carrying the instructions from DNA by controlling the synthesis of protein

Question 6

DNA

Answer 6

Portions of DNA are called genes.
DNA is tightly wound into chromosomes and located in the nucleus of cells.
DNA cannot leave the nucleus.
DNA is DOUBLE STRANDED(2 sided)

Question 7

RNA

Answer 7

RNA is SINGLE STRANDED and does not have to stay in the nucleus

RNA is not found in chromosomes because it does not carry the genetic code, however it can read the DNA code and take the information out of the nucleus.

RNA’s main job is to build proteins
Virus with RNA only referred to as Retrovirus and the human retrovirus is HIV 1 and HIV 2 responsible for AIDS

Question 8

Applications of PCR/ Molecular Based Testing in Clinical Microbiology

Answer 8

Rapid or high-throughput identification of microorganisms
3-4 hours for the results to be reported.
Detection and analysis of resistance genes
Genotyping
Classification
Discovery of new microorganisms

Question 9

Target Microorganisms for PCR Based Testing

Answer 9

Those that are difficult or time-consuming to isolate
e.g., Mycobacteria, SARS
Hazardous organisms
e.g., Fungi - Histoplasma, Coccidiodes
Those without reliable testing methods
e.g., Virus - HIV, HCV ,
High-volume tests
e.g., Bacteria - S. pyogenes, N. gonorrhoeae, C. trachomatis, MRSA

Question 10

How does PCR work

Answer 10

By mimicking DNA synthesis in cells
Requires only trace amounts of DNA as starting material for a PCR reaction
PCR helps make multiple copies of a specific sequence (or gene) of interest
Once the PCR amplification is done, the amplified DNA sequences are compared to nucleotide (DNA) sequences from known sources such as specific person, animal, bacteria, virus

Question 11

Thermocycler

Answer 11

PCR is performed in a thermocycler
A thermocycler can heat and cool quickly, accommodating the temperature shifts during PCR steps
Cetus corporation developed the first thermal cycler named ‘Mr Cycle’

Question 12

Real Time - PCR

Answer 12

Real Time PCR is designed to detect and measure RNA
PCR amplifies DNA, many viruses utilize RNA as their genetic material
In real time PCR, the RNA strand is first converted into a DNA strand by using an enzyme called reverse transcriptase
Once the DNA strand is formed, it undergoes the same amplification as in PCR but uses a fluorescent dye attached to either the polymerase.

Question 13

Advantages of PCR Detection of Microorganism Resistance to Antimicrobial Agents

Answer 13

Mutated genes are strong evidence of resistance to antibiotics
Stat detection of organisms without culturing especially critical during an epidemic outbreak of an infectious disease
Direct comparison of multiple isolates in epidemiological investigations (Pandemics)

Question 14

Pharmacogenetics for Cancer

future of PCR – Personalized Medicine

Answer 14

Provide real-time decision support
Sequences the tumor and determine what drugs are effective
Identifies the gene in the tumor and determines the prognosis and the survival of the patient to the cancer
More targeted medicines (“personalized medicine”)
More effective
Safer
More cost-effective
Based on a better understanding of inter-individual differences among patients

Facilitates individualized drug therapy to maximize efficacy, minimize adverse drug reactions, and reduce health care costs

Question 15

DNA Fingerprinting Uses

Answer 15

Paternity testing
Identification of criminals (e.g. murderers, rapists, letter bombers)
Identification of deceased individuals with mutilated or decomposed bodies (e.g., the military, 9/11 victims)