Happy Monday everyone!
In a previous post I did an inspectional reading of the book “Principles of Gene Manipulation and Genomics” in honor of my order from the Odin, “The DIY Genetic Engineering Home Lab Kit”, being updated. Well good news, IT HAS ARRIVED! In the previous post I stated that my goal was to finish part 1 by the time the equipment arrives. I did not, but I do figure it would be fun to share my notes on what’s been covered thus far.
Some of the information that may be relevant to you (explanations of PCR or Electrophoresis) were not initially taken in my notes since I am already familiar with them.
Chapter 2 – Basic Techniques: (Chapter 1 was covered in the inspectional reading)
(The primary techniques covered in this chapter are Electrophoresis, PCR, techniques for transforming E-Coli, and blotting so separated DNA can be worked with)
DNA Changes shape during electrophoresis, switching between a ball + string Pulled Electrophoresis from different, pulsed electrophoresis from different directions can jiggle the DNA and allow larger sections of DNA to undergo electrophoresis.
Blotting exists to move DNA, RNA or Proteins separated by gel electrophoresis onto a fixed membrane that you can do tests with. It frequently uses capillary action to suck the fluid containing the separated DNA upwards.
Southern blotting is for DNA, northern is for RNA and Western blotting is for proteins. The nature of the membrane may be altered based on what it’s binding.
“Probes” can be made by making a complimentary sequence to the one you’re looking for + tagging it radiologically.
For DNA staining scientists switched from ethidium bromide to SYBRSafe upon finding out the former is a carcinogen. (I’ll look up how to do this).
Techniques for transformation of (successfully putting foreign DNA into) E-Coli:
– It can take up plasmids
– Will take up DNA from bacteriophage λ when in a solution of CaCl2.
– Will take DNA when chilled with CaCl2 + trace minerals, then given a “heat shock”
– Also works with electroporation
The stringency of probe-blot hybridization is controlled primarily by the salt content and temperature of the post-hybridization wash.
Skipping PCR due to familiarity. I will likely do special posts on electrophoresis and PCR later. However, this book explains it rather well. In the meantime, I highly recommend this video on Youtube for PCR and this video on Youtube for electrophoresis.
Chapter 3 – Cutting and Joining DNA Molecules:
(borrowed from inspectional reading) Ch. 3 - Cutting and Joining DNA Molecules. - Cutting DNA molucules lists several points about restriction endonucleases and contributing factors which may enable or impede their function in cutting DNA. - Cites DNA Ligase as key for joining molucules "in vitro" - Talks about matching the correct ends for ligase to join and techniques for that. - Also cites alternative (but evidently less preferred) method of joining DNA. (end borrowed)
Before the 1960’s the only method for breaking up DNA was by mechanical shearing. This could be done by throwing it in a blender or subjecting it to ultrasonic blasts, while some had relative advantages in controlling the average size of the fragments, everything else about the process was random. In the 1960’s phage biologists discovered K12 restriction endonuclease in E. Coli that cuts DNA into “Large discrete fragments.” From there people figured it was targeting a particular sequence. However, it would still cut several kilobases away (somewhat randomly) from it’s target. The first usable restriction endonuclease was discovered in the 1970’s. (Side note, restriction endonucleases are proteins that cut up sections of DNA which it suspects are of viral origin in order to prevent the viral DNA from executing it’s code in the host genome)
Understanding restriction endonucleases is important because they can be useful in cutting and joining DNA for you, and can also be dangerous for your plasmid if your custom dna accidentally contains a target sequence.
When two strains of E Coli (C and K) are plated and infected with phage lambda, the success of the phage depends on the previously infected strain. If it infects C first, then K, it gets mostly restricted. If it infects C then C, or K then K, then on the second pass it infects without problems. This is because target sequences for the restriction endonucleases do exist in the bacteria’s genome, but those sites are methylated to tell the endonuclease to spare them. Once the phage’s DNA passes through that strain successfully, the new dna is manufactured by that strain and receives methylation at the restriction sequences.
There are four types of restriction nuclease, Type I, Type II, Type III and Type IIs (why can’t biologists count!?)
Restriction endonucleases have a naming scheme:
First letter of species first name. Capital, italics
First two letters of speciaes last name, italics
If a particular strand, letter of that strand
Roman numerals of number of nuclease (is it the i’th one to be discovered, the ii’th one, or the v’th one)?
For Homing endonucleases, there is a prefix I for introns and PI for inteins
Where restriction or modification are relevant, R or M as prefixes are also used.
Information on restriction enzymes are likely stored on a database. They can probably also be purchased once you know which one you need.
Isoschisomers = restriction enzymes that recognize the same sequence and cut at the same place
Neoschisomers = restriction enzymes that recognize the same sequence but cut at different places.
Star activity = willingness for an endonuclease to accept a wider variety of sequences with increased temperature or pH.
Restriction endonucleases are affected by the relative prevalence of GC/AT pairs. Uneven pairs can lead to a less random availability of restriction sites.
Also, not all restriction sites (even the same sequence) are cleaved evenly. In phage lambda, the ones near the 5′ end are cleaved the most. Also, different endonucleases that recognize the same sequence may not be equally sensitive to methylation.
The enzyme that joins different DNA molecules is called DNA Ligase.
Linkers have blunt ends but adaptors do not. Both are used for connecting multiple restriction sites.
PCR usually modifies the ends of the DNA being copied, which means that special provisions need to be made for cloning. One way to address this is to include a restriction site in the piece of DNA to be amplified, plus a few base pairs of DNA on the end to act as a buffer to modification.
Topoisomerase can both cut and join (so endonuclease and ligase are not necessary). It is better than other restriction enzymes in combination with PCR and can do in 5 minutes what it takes the ligase to do overnight.
Site specific recombinases (Cre, Flp and Int lambda) can be used for cloning and integrating DNA from much larger and very specific restriction sites, and Flp can clone DNA in such a way as it can be done multiple times (as in passing to phage lambda, to put it in e. coli, to put it into another phage, to put it in a plant pathogen, to insert it into plants), whereas other methods risk modifying the DNA along the way (including methyl group tagging, not strictly modification to the source code).
Chapter 4 – Basic Biology of Plasmid + Phage Vectors:
The span/variety of bacterial hosts that can be occupied by a particular plasmid depends on which and how many of the proteins needed to replicate the plasmid are stored on the plasmid itself. A plasmid that carries all or almost all genes to replicate itself is viable in a much larger variety of bacterial hosts. (Note: plasmids are mostly circles of DNA that can be taken up by a bacteria and easily copied and shared with other bacteria that sits outside of their genome. This is essential for bacteria since they divide clonally and use plasmids as a form of gene-transfer/sharing in lieu of sexual reproduction).
The number of copies of a single plasmid that can take place inside of a bacteria (called the “copy number”) is controlled by the plasmid’s replication machinery. Ex. Rep A is a necessary protein to replicate it’s host plasmid, but it also has the capacity to bind to the gene sequence encoding Rep A. If too much RepA is produced, they bind to the their own source genes and prevent more RepA from being produced. Because of these types of restrictions, two different plasmids that use the same replication machinery will not be able to exist in the same cell, one will shut the other down.
(Did not complete chapter 4, notes to be continued at a later time)