MOL BIO LAB MIDTERMS - PCR

Taq polymerase

The most commonly used polymerase and important part of buffer/reagent. A heat stable DNA form of polymerase.

Thermus aquaticus

Taq polymerase is taken in this bacteria

Polymerase Chain Reaction

A multiplication of billions copies of the gene target. Production of millions or even billions of copies of a DNA molecule in just a few hours.

Kary Mulis and Michael smith

Developers of PCR. They won nobel prize in chemistry 1993.

Detects DNA sequences

Diagnose genetic diseases

Carries out DNA fingerprinting

Detects bacteria or viruses

Determines the molecular phylogenetics of organisms

Applications of PCR

140 to 200 uL

PCR volume for extraction

Maximum of 2 hours

Extraction of nucleic acids turn around time

Blood

Specimen sample used in genetic testing

Plasma

Specimen sample used in virus detection

Swab

Specimen sample used in microorganism

Tissue biopsy

Specimen sample used in cancer

Automated extraction (Roche's MagNA pure, Promega, Qiagen)

This extraction method is commonly used in clinical labs. It has high volume testing and higher capital costs.

Manual extraction (Qiagen kit)

Typically uses commercial kits. It has low volume testing, low capital cost. It is often used in research labs.

Phenol and choloroform

Organic DNA isolation uses these substances

High salt and precipitation

Inorganic DNA isolation uses these substances

Magnetic silica beads
Columns

Solid-Phase DNA isolation uses these methods

Fragile

Working with RNA is hard because it is?

RNase Zap

RNases easily degrade RNA. Before working with RNA you need to put this to rapidly inactivate RNase

Screening Phase

Phase of PCR where during the first few cycles the appropriate part of the template is selected by specific binding and extension of the primers

Amplification phase

Phase of PCR where during the subsequent rounds of PCR, the copy number of the desired sequence increases exponentially.

Primer design

This should be checked against available sequence databases to ensure that they are homologous to the desired region

Denaturation

Step of PCR where the separation of the strands happen

Annealing

Step of PCR where the primer and template sticks together

Extension / Elongation

Step in PCR where the new strand grow in length

Denaturation
Annealing
Extension or Elongation

Step of PCR

90 to 95

Temperature of denaturation

40 to 60

Temperature of annealing

70 to 75

Temperature of extention/elongation.

17 to 24 bases

Primer design length

40% to 60%

Primer design GC content

G or C

Primer should preferably end in this base pairs

Real time PCR

Other types of PCR that has a way of quantifying the amplification of DNA as it occurs real time

Quantitative reverse transcription PCR

Other types of PCR where starts with the reverse transcription of either total RNA or polyA+ RNA into cDNA using a reverse transcriptase