BIO203 Current Solved Paper

Bio203 date 2 January

Microbiology application (long)

PCR is the most commonly used nucleic acid amplification technique for the diagnosis of infectious disease, surpassing the probe and signal amplification methods. The PCR can amplify DNA or, when preceded by a reverse transcription (RT) incubation at 42-55 °C, RNA.

Forensic application (long)

The most widely used application of PCR in forensic labs is the amplification of short tandem repeat (STR) loci used in DNA typing. The STRs are routinely evaluated in concert with 16 or more reactions, a multiplex, run in one test tube simultaneously. Deoxynucleoside triphosphates (dNTPs), the substrates for DNA polymerizing enzymes, have long been known to be limited in their concentration in cells because the enzyme that synthesizes deoxynucleotides from ribonucleotides, ribonucleotide reductase (RNR), is synthesized and enzymatically activated as cells enter th

dNTPs (short)

DNA polymerases use dNTPs to extend a DNA primer hybridized to a complementary DNA template. As the appropriate dNTP is incorporated into the growing strand, a phosphodiester bond is formed between the 3′-hydroxyl terminus on the growing strand and the α-phosphate of the incoming dNTP

nested and multiplx (short)

Nested PCR usually involves two sequential amplification reactions, each of which uses a different pair of primers. The product of the first amplification reaction is used as the template for the second PCR, which is primed by oligonucleotides that are placed internal to the first primer pair.

What is the difference between nested PCR and conventional PCR?

Conventional PCR (C-PCR) has been used to detect specific target genes in various microorganisms (5, 6, 13). Nested PCR (N-PCR) was developed to improve sensitivity but can give erroneous positive results due to DNA contamination (1). Multiplex PCR is a variant of PCR which enables simultaneous amplification of many Targets of interest in one reaction by using more than one pair of primers. Multiplex-PCR consists of multiple primer sets within a single PCR mixture to produce Amplicons of varying sizes that are specific to different DNA sequences.

Hot start long

THREE STEPS of PCR

I.Denaturation II.Annealing III. Extension

INITIALIZATION STEP

•  This step consists of heating the reaction to a temperature of 93°C – 96°C, which is held

For 1-10 minutes.

DENATURATION

•  First regular cycling event and consists of heating the reaction to 93°C – 98°C for 20- 45

Seconds.

•  It causes melting of DNA template yielding single strands of DNA.

ANNEALING

•  The reaction temperature is lowered to 40-65°C for 20-40 seconds.
•  Annealing of the primers to the singlestranded DNA template.

EXTENSION

•  The temperature at this step depends on the type of DNA polymerase used.
•  Taq polymerase has its optimum activity temperature at 70- 80°C.
•  Commonly 72°C is used.
•  At this step the DNA polymerase synthesizes a new DNA strand complementary to the

DNA template strand by adding dNTP’s

BLANK REACTION

•  Controls for contamination contains all reagents except DNA template.

NEGATIVE CONTROL

•  Controls for specificity of the amplification reaction contains all reagents and a DNA

Template lacking the target sequence.

CONCLUSION

•  The PCR product should be of the expected size.

IN SITU PCR

•  In Situ PCR (ISH ) is a polymerase chain reaction that actually takes place inside the cell On a slide.