Molecular Techniques Flashcards
Explain the principles of nucleic extraction
There are three basic steps: cell disruption, separation of DNA and, concentration of DNA
Calculate the concentration of RNA or DNA
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
Calculate and assess nucleic acid purity
Analyzing the A260:A280 ratio
pure dsDNA: 1.7-1.9
pure RNA: 2.0
List the reagents used in end point PCR
template DNA, DNA polymerase, dNTPs, PCR buffer, primers, MgCl2
Explain and describe the denaturation step used in end point PCR
occurs at 92-95C for 10-30s
breaks the bonds holding together two strands of DNA
Describe reverse transcription PCR
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
Describe no RT control
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
Explain the principles of nucleic acid separation using agarose gel electrophoresis
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
Explain the principles of nucleic acid separation using capillary gel electrophoresis
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.
Explain Sanger Sequencing
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
Explain the principles of real-time PCR
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
Describe and compare dual hybridization probes and hydrolysis probes
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
Describe the no template PCR control
contains all the components for PCR but the nucleic acid portion is replaced for water. This ensures there is no contamination
Describe the negative PCR control
a nucleic acid sample in which the target is not present. This validates the specificity of the reaction
Describe the positive PCR control
has a target nucleic acid of sufficient quantity and purity. Ensures that the PCR conditions were sufficient to amplify
Describe the internal PCR control
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
Explain transcription mediated amplification
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
What are the two different extraction methods
liquid: phenol or chloroform
solid: magnetic silica beads
What are possible reagents in a lysis solution
chaotropic salts, detergents, alkaline denaturant and, proteases
Explain and describe the annealing step used in end point PCR
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
What are examples of chaotropic salts
guanidinium isothiocyanate, urea and, sodium dodecyl sulphate
What are possible reagents in a lysis solution
chaotropic salts, detergents, alkaline denaturant and, proteases
What are the two different extraction methods
liquid: phenol or chloroform
solid: magnetic silica beads
How does the qiagen symphony work
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
Explain the function of template DNA in PCR
the gene that serves as the template for amplification
What are examples of chaotropic salts
guanidinium isothiocyanate, urea and, sodium dodecyl sulphate
Explain and describe the extension step used in end point PCR
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
Explain the function of DNA polymerase in endpoint PCR
the enzyme replicates the template DNA
Explain the function of dNTPS in end point PCR
provides the building blocks for the new DNA
What are examples of chaotropic salts
guanidinium isothiocyanate, urea and, sodium dodecyl sulphate
Explain the function of template DNA in PCR
the gene that serves as the template for amplification
Explain the function of PCR buffer in PCR
to keep the polymerase at the optimal pH for reaction
Explain the function of the primers in PCR
the primer is complimentary to a region either upstream or downstream of the target DNA. Taq recognizes the 3’ end and binds
Explain the function of MgCl2 in PCR
it is a cofactor for Taq polymerase activity
What is ethidium bromide
an intercalating dye that binds between complementary base pairs of dsDNA
