Define Molecular Diagnostics
Use of DNA, RNA and protein aka amino acid, to test for specific disease
What is one of the way molecular diagnostic
... [Show More] are use in the lab and gave an example?
To speed up the identification of
1) slow growing microrganism
2) Fastidious microrganisms
3) Microrganism that wont grow on conventional media>
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What are the Molecular diagnostic Principle and describle each one?
1) Target sequence, which is to target the a base sequence for that a single specific mircoorganism.
2) Amplification which is the replication of the target sequence that we are after.
3) Detection is use to detect that we have the right sequence after all.
Describe DNA transcription
The process through which DNA sequence is enzymatically copied by RNA polymerase to produce a complemetary RNA, aka messager RNA (mRNA)
Describe the structure of DNA.
1) It is a double helix
2) Sugar
3) Phosphate group
4) Base
What are the base in a nucleotide? Which one are Purine and which ones are Pyrimidines?
1) Purine are Adenine, and Guaine
2) Pyrimidines are Cytosine and Thymine
What are the bond between
1) nucleotides
2) 2 strands of DNA
1) Phosphodiester, which are strong covalent bond
2) Hydrogen bonds which are unstable.
What does it mean by the DNA are Antiparallel nature?
It means that there is one strand that read form 5' to 3' and second strand read form 3'to 5'.
What is unwind frist DNA replication and what is use to stablize it?
1) The helicase are unwind by DNA Gyrase
2) Protein help stablizie the unwound DNA
The leading strand is synthesizes, how?
1) DNA polymerase III is use to contiinuously synthesizes form 5' to 3'.
The lagging strand is synthesizes, how?
Synthesize is dicontinuously by
1) RNA polymerase make RNA primer
2) RNA primer then extend by DNA polymerase to make the Okazaki Fragment
3) Then DNA polymerase I and replace the RNA primer with DNA
4) DNA ligase join the Okazaki Fragemnt to the growing strand.
1) How is the parent strand of DNA read?
2) In what direction is the daughter strand synthesized?
1) 3' to 5'
2) 5' to 3'
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What does DNA Gyrase do in DNA replication?
1) Unwind the helix
2) add protein stabilize
What does RNA Polymerase do in DNA replication?
They make short RNA primers
What does DNA Polymerase III do in DNA replication?
This add bases
What does DNA Polymerase I do in DNA replication?
1) It digest the RNA primer
2) Then it add the base
What does the DNA ligase do in DNA replication?
It add the DNA phosphodiester bonds
What is the result of PCR (polymerase chain reaction)?
1) Technique that result in Nucleic Acid Amplifiaction of a DNA region.
What is the product of PCR (polymerase chain reaction) and why is called that?
1) Amplicons
2) During amplification where you take one DNA segment and make a millions copies.
What is need for amplifications during a PCR in-vitro?
1) Enzymes like polymerase
2) Primers
3) Master Mix
4) Need to do a series of heating and a series of cooling.
In PCR, what reagents does the master mix contain and why?
1) Tag (thermus aquatice) which act like DNA polymerase.
2) Deoxynucleoside triphosphate (dNTP) free nucelociteds
3) Primers which give a starting point
4) Buffer and MgCl2 which is use to maintain the pH and Mg 2+ is Cofactor
5) Mineral oil or PCR Gems which subsited by lipid heat. These help prevent contamination and evapulation.
What are the Requirements to perform a PCR?
1) Specimen form either a Primary specimen or sub culture
2) Special Area so you can limit some of the contamination because if you get a few bacterial they will turn into millions.
3) Thermocycle
In general, what is the PCR Produce?
1) This is the begin of a cycle, you heat up the specimen to 95 degree C for 15 to 30 sec.
2) then you cool down the specimen to 55 degree C for 30 sec to 120 sec.
3) You heat up the specimen to 72 degree and this is the end of the cycle.
4) Then you repeat, over and over and over...
In the PCR Procedure for step 1,
1) What is the purpose of this step?
2) What is the temperature set at in this set?
3) How long do we incubate this step for?
1) Denaturation step, which take the double helix and split them up into two single strand of DNA. Also this is the begin of a cycle and you add the master mix.
2) 95 degree C
3) 15 to 30 sec
In the PCR Procedure for step 2,
1) What is the purpose of this step?
2) What is the temperature set at in this set?
3) How long do we incubate this step for?
1) Annealing step, which is where the attachment of the primer happen.
2) Cool it down to 55 degree C
3) 30 to 120 secs
In the PCR Procedure for step 3,
1) What is the purpose of this step?
2) What is the temperature set at in this set?
3) How long do we incubate this step for?
1) Extension step, which is where the polymerase add the base to the DNA. This is also the end of a cycle. Remember that polymerase code form 5 to 3.
2) 72 degree C
3) 1 to 2 minutes.
In the PCR, how many cycle does it take to get the Target Region?
1) Cycle 3 in order to 2 target region.
What are the methods that PCR use?
1) Gel Electrophories
2) Fluorescence
3) Chemiluminescence
How does fluroescence work in detecting amlicon PCR?
1) The tag Amplicon / Label Amplicon own works it Nucleotide is in the DNA chain.
2) So free target nucleotide do not work.
3) Shorter wavelength hit the flourescence dye which make a longer wavelength.
For Chemiluninescence in the PCR,
1) What can measure it?
2) What is need for it to work?
1) It can be measure by a chemiluminometer or Luminonmeter
2) You need another Dye for it to work.
What is same difference between the first generation of PCR verus the Real-time PCR aka 2nd generation?
1) Amplicons are assayed as they accumulate.
2) use Florochrome
3) UV light source inside the Thermocycler
4) Camera connected to a computer system.
What are the advantage of using a Real-time PCR (2nd generation)?
1) Quicker
2) Less chance of contamination
3) Less hazardous waste
Describe the procedure of DNA probes?
1) You denaturation of the DNA
2) Hybridization with label DNA, the probes, during incubation
3) Add Reagent to digest any unhybridized probes
4) Separation of hybriduzed DNA by using a magnet. The Hybridized DNA stick to the wall of the tube.
5) Removal of digested unhybridized probe by washing.
6) You measure it by Chemiluminometer. [Show Less]