Lab 5 Flashcards
What is 16s rDNA and how is it used to identify sepcies of bacteria
6sRNA is the piece of DNA in bacteria that codes for the small subunit of ribosomal RNA
Different bacterial species have unique 16s rDNA sequences
You can match a sample sequence agaisnt a database of all known 16s rDNA sequences to identify a sample
What are proteolytic enzymes necessary for rDNA sequencing
lysozyme digests the peptidoglycan cell wall + digestive buffer
grants acess to the interior of the cell
Why do you need to deactive the proteolytic enzymes used in a cell wall digest
since they are protyolytic, they would digest the other enzymes that we will be using
we can heat deactivate these enzymes at 100C for 3 hrs
After you spin the sample in the centrifuge, what is the pellet and what is the supernatent?
Where is the DNA?
the cellular debris is spun down in the centrifuge and appears as a solid deposit (pellet) at the bottom of the tube. The DNA is contained in the supernatant (the liquid), which is then transferred to the PCR tube.
What is PCR?
PCR (Polymerase Chain Reaction) is a technique that allows many copies of DNA to be made
In PCR, single-stranded DNA is made by heating a chromosome fragment to 95°C. It is then cooled so that the primers anneal to the original DNA strands and DNA polymerase can bind and copy each strand. With repeated heating and cooling, millions and even billions of copies of DNA can be made in a few hours.
The discovery of thermotolerant polymerase
the discovery of chemoautotrophic bacteria that live in the hydrothermal vents in the deep ocean floor. These creatures live in the vents where temperatures exceed 100°C; consequently, their DNA polymerases remain functional at such temperatures that would have denatured an ordinary creature’s DNA polymerases. DNA polymerases isolated from these bacteria have made it possible to develop automatic thermocycler machines without the need to add new polymerases each cycle.
What are primers, and why are they added to PCR
Primers specifically bind to regions flanking the 16S rRNA gene
Bind to 16s rDNA and initate the replication process
PCR master mix
The PCR Master Mix solution (in the red-capped bottle) contains the following: water; a buffer to keep the mixture at the correct pH for the PCR reaction; large quantities of the four nucleotides adenine, cytosine, guanine, and thymine; large quantities of oligonucleotide DNA primers that bind the 16S rDNA region to initiate the replication process; and a heat-stable DNA polymerase that extends the copy DNA strand.
PCR positive control
contains positive control DNA (the solution of 16S rDNA in the green-capped bottle)
=master mix
PCR negative control
negative control reaction contains sterile deionized water
= master mix
Why are highly conserved sequences important for DNA sequencing
Highly conserved regions are some parts of a gene that are extremely similar among different species. They are important because universal primers bind to them so that they can be used to copy DNA from a variety species of bacteria.
What are the three steps of PCR cycling
Initial incubation step: 95°C for 10 minutes
30 cycles of the following sequence of steps:
Melt: 95°C 30 seconds
Anneal: 60°C 30 seconds
Extend: 72°C 45 seconds
Final extension step: 72°C 10 minutes
Final step: 4°C store at this temperature
During each cycle, the first step (melt) is to separate the two DNA chains in the double helix by heating the vial containing the PCR reaction mixture to 95°C for 30 seconds. The primers cannot bind to the DNA strands at such a high temperature, so the vial is cooled to 60°C. At this temperature, the primers bind (anneal) to the single-stranded DNA. (The reason the two separated strands of DNA do not reanneal is that there is a large excess of primers in the solution; therefore, it’s more likely for the DNA strands to bind to the primers instead of to each other.) The final step (Extend) is to allow the DNA polymerase to extend the copy DNA strand by raising the temperature to 70°C for 45 seconds.
Why is it useful to run a gel after amplification
the gel should contain three lanes: one for the negative control (i.e., water), which should not have a product unless the water was contaminated; another for positive control (PCR product of a known DNA sequence) to make sure that the PCR itself worked; and the last lane for your sample.
Running a gel is actually one method of purification. Once the PCR product is in the gel, you can cut out the band corresponding to the PCR product and isolate the DNA from the gel.
Micro-concentrator colum for POCR purification
Final product
DNA is trapped in the column, need to get it out.
invert tube, add buffer and spin again (discard first supernatent)
centrifuge again, and discard the column, keep the collected liquid
The final collection tube should now have many pieces of 1,500bp-long 16S rDNA, with a very small amount of longer DNA strands (which are contaminants).
What is the purpose of the second PCR
PCR cycle sequencing
-> terminates at random pts with a fluorescent marker
multiple primers are used (12) to get overlapping sequences
