Molecular Techniques

Question 1

Explain the principles of nucleic extraction

Answer 1

There are three basic steps: cell disruption, separation of DNA and, concentration of DNA

Question 2

Calculate the concentration of RNA or DNA

Answer 2

50 ug/mL of dsDNA has a A260 of 1.000

33 ug/mL of ssDNA has a A260 of 1.000

40 ug/mL of RNA has a A260 of 1.000

Question 3

Calculate and assess nucleic acid purity

Answer 3

Analyzing the A260:A280 ratio

pure dsDNA: 1.7-1.9

pure RNA: 2.0

Question 4

List the reagents used in end point PCR

Answer 4

template DNA, DNA polymerase, dNTPs, PCR buffer, primers, MgCl2

Question 5

Explain and describe the denaturation step used in end point PCR

Answer 5

occurs at 92-95C for 10-30s

breaks the bonds holding together two strands of DNA

Question 6

Describe reverse transcription PCR

Answer 6

uses RNA as a template instead of DNA. RNA is converted to cDNA using reverse transcriptase. It can be completed in one step or two

one step: cDNA and PCR are done in the same tube

two step: cDNA is made and then moved to another tube for PCR

Question 7

Describe no RT control

Answer 7

this control is set up at the transcription step of RT-PCR. Reverse transcriptase is omitted from the reaction, no DNA should be detected at the end of the assay

Question 8

Explain the principles of nucleic acid separation using agarose gel electrophoresis

Answer 8

agarose creates a network of pores that DNA must fit through. A buffer carries an electrical charge from the cathode to the anode. The DNA moves with the charge, smaller fragments travel further as they get caught in the pores less. DNA is visualized on the gel using special dyes (sybr green or ethidium bromide). Samples are compared to a molecular ladder to determine fragment sizes

Question 9

Explain the principles of nucleic acid separation using capillary gel electrophoresis

Answer 9

occurs within a long, thin silica tube with a polyimide coating. A small volume of sample is injected via electrokinetic injection. DNA migrates, separating based on size and is detected via the florescent markers that have been attached.

Question 10

Explain Sanger Sequencing

Answer 10

ddNTPs are bound with a florescent marker, each different colour marks a different nucleotide. When a ddNTP binds, replication terminates. DNA strands of various lengths are created and then they are separated using capillary electrophoresis

Question 11

Explain the principles of real-time PCR

Answer 11

qPCR products are labelled with fluorescence allowing for detection of products without having to use electrophoresis. This cuts down on time, increases sensitivity and reduces risk of contamination

Question 12

Describe and compare dual hybridization probes and hydrolysis probes

Answer 12

hydrolysis: probe is an oligonucleotide complimentary to the target sequence. The probe is modified with a fluorophore attached to the 5’ end. Intact probe does not fluorescence. Taq polymerase’s 5’-3’ exonuclease activity cleaves the probe which causes fluorescence this occurs during elongation. Fluorescence is proportional to PCR product

dual hybridization: two labelled oligonucleotides that bind in a head to tail fashion are used. When they get close they fluorescence, which occurs during hybridization. Fluorescence is proportional to PCR product

Question 13

Describe the no template PCR control

Answer 13

contains all the components for PCR but the nucleic acid portion is replaced for water. This ensures there is no contamination

Question 14

Describe the negative PCR control

Answer 14

a nucleic acid sample in which the target is not present. This validates the specificity of the reaction

Question 15

Describe the positive PCR control

Answer 15

has a target nucleic acid of sufficient quantity and purity. Ensures that the PCR conditions were sufficient to amplify

Question 16

Describe the internal PCR control

Answer 16

simultaneously extracted and amplified, something that is known to be there but unrelated to the target gene (ex. BCR). Ensures there are no issues in the entire process

Question 17

Explain transcription mediated amplification

Answer 17

used to detect chlamydia and gonorrhoeae the assay detects RNA. Prokaryotic organisms are lysed in a buffer. RNA is then captured by a oligo that hybridizes to a magnetic particle. This allows the RNA to be immobilized and washed. The oligo is also the primer for reverse transcriptase. After the creation of cDNA, DNA is created and then PCR continues as normal using a second oligo as the primer for RNA polymerase. The end product is RNA

Question 18

What are the two different extraction methods

Answer 18

liquid: phenol or chloroform

solid: magnetic silica beads

Question 19

What are possible reagents in a lysis solution

Answer 19

chaotropic salts, detergents, alkaline denaturant and, proteases

Question 20

Explain and describe the annealing step used in end point PCR

Answer 20

occurs at 50-60C for 30s

temperature is decided using the Tm-5C, Tm is calculated by: 4(G+C)+2(A+T)

primers and DNA polymerase bind to the 3’ end of the ssDNA

Question 21

What are examples of chaotropic salts

Answer 21

guanidinium isothiocyanate, urea and, sodium dodecyl sulphate

Question 22

What are possible reagents in a lysis solution

Answer 22

chaotropic salts, detergents, alkaline denaturant and, proteases

Question 23

What are the two different extraction methods

Answer 23

liquid: phenol or chloroform

solid: magnetic silica beads

Question 24

How does the qiagen symphony work

Answer 24

samples are incubated in a lysis buffer containing a chaotropic salt and proteinase K. Cells lyse and cellular components are released. Magnetic silica beads are added and the nucleic acids bind then a magnetic rod transfers the nucleic acid material to a series of reaction vessels where DNA is washed and eluted

Question 25

Explain the function of template DNA in PCR

Answer 25

the gene that serves as the template for amplification

Question 25

What are examples of chaotropic salts

Answer 25

guanidinium isothiocyanate, urea and, sodium dodecyl sulphate

Question 25

Explain and describe the extension step used in end point PCR

Answer 25

occurs at 72C for 30s

polymerase forms dsDNA by adding complementary deoxynucleotides

general rule is 1 min/1 kbase and 30s for anything less than 1kbase. Temperature is based on the polymerase used

Question 25

Explain the function of DNA polymerase in endpoint PCR

Answer 25

the enzyme replicates the template DNA

Question 25

Explain the function of dNTPS in end point PCR

Answer 25

provides the building blocks for the new DNA

Question 25

What are examples of chaotropic salts

Answer 25

guanidinium isothiocyanate, urea and, sodium dodecyl sulphate

Question 25

Explain the function of template DNA in PCR

Answer 25

the gene that serves as the template for amplification

Question 25

Explain the function of PCR buffer in PCR

Answer 25

to keep the polymerase at the optimal pH for reaction

Question 25

Explain the function of the primers in PCR

Answer 25

the primer is complimentary to a region either upstream or downstream of the target DNA. Taq recognizes the 3’ end and binds

Question 25

Explain the function of MgCl2 in PCR

Answer 25

it is a cofactor for Taq polymerase activity

Question 26

What is ethidium bromide

Answer 26

an intercalating dye that binds between complementary base pairs of dsDNA