A note before we begin, genetics is a massive field which ideally requires a degree to tinker with. No amount of information that can be provided on this wiki is equivalent to the amount of practice and time in the lab. This will serve as the basics.
All life runs on DNA, genetic engineering is the process by which we study the current genetic makeup of an organism, either by sequencing it's genome or various other techniques, and then modify that sequence in one way or another. This process is different depending on what you're hoping to modify. For example plants are orders of magnitude easier to modify than animals. This is because anything with moving parts has to be significantly more complicated to support itself and defend itself. All DNA is composed of 4 letters (it changes for RNA but ignore that for now) A, T, C and G. A always pairs with T and C always with G. Each piece of DNA is double stranded and one strand is the mirror of the other. If strand 1 is AATTGGCC then strand 2 would read TTAACCGG. However each strand runs in opposite directions. At one end of DNA is OH group that marks the end of the strand. This is known as the 3 prime end (3'). the other end, marked by a phosphate group is known as the 5 prime end (5'). When DNA is being synthesized it is Almost always synthesized in the 5' to 3' direction and as such it is said that DNA runs 5' to 3'. This is the bare basics. This does not cover replication or much else. For your reading pleasure I would suggest looking up DNA replication, Mitosis, Meiosis, leading strand, lagging strand, DNA polymerase (1 and 3), DNA transcription, DNA translation Protein synthesis, ribosomes, the basics of the cell, post translation modification of proteins, Regulation mechanisms of DNA (operators, enhancers, primers, sigma factors, etc). As you read this ought to point you in the direction of any other bits you're missing before continuing below.
Forget what the professor in second year taught you. Punnet squares are not what we need here. No breeding and backcrosses and test crosses and pedigrees. If you'd like to learn about all that, Youtube is your friend but it's useless to grinders.
For us sequencing is your friend. It is a group of processes that allow one to take an isolated piece of DNA and be presented with its exact chemical code. A note here, sequencing in all of it's forms is *expensive*. If you're lucky, the thing you want to work with has already been sequenced.
There are a large number of ways things can be sequenced, and the one you choose depends on how much money you feel like spending and the length of the bit of DNA you need sequenced.
Sanger sequencing (the old school method(read length 800bp))
It's great for short sequences and was the original method for sequencing. The process is fairly simple. Take your fragment of DNA, say some new gene you're working on. Run it through PCR (see below) so you have a whole bunch of it. Put a little bit in 4 small vials along with buffer and an RNA primer that matches the start of the sequence. Add in 1 type of dideoxy nucleotide to each vial (ddA in the first ddT in the second etc.) Then add all four of the regular nucleotides to all 4 vials. Finally add in DNA polymerase. What happens is DNA polymerase begins to synthesis a matching piece of DNA but when it uses a dideoxy nucleotide instead of the regular ones it gets stuck and stops leaving a short fragment. You then take each fo the four vials and load the contents of each into a separate well of an electrophoresis gel. Run the gel and the add a stain to show you the bands that form. By reading the bands from top to bottom you'll have your sequence. If this all seems to complicated it is because reading about it makes it sound like a nightmare. It's much simpler in video form.
Polymerase Chain Reaction is a method of multiplying specific segments of DNA.
The requirements are: *source DNA material (RNA can be used if it is transcribed to DNA first)
- Nucleotides The ATCG building blocks
- Polymerase - the enzyme that will add the ATCG to the original strand. The Enzyme is thermally stable, that is to say that it won't be destroyed at the high denaturing temperature.This will be the most expensive component of your reaction.
- Buffer - contains salts and ions to facilitate and stabilise the reaction.
- Primers - possibly the most important part of the reaction. (see below)
Basic Primer Design - PCR reactions require primers which are a short (up to 20BP) synthetic sequences of complementary DNA to the area you want to amplify.
So say you wanted to amplify GENE123 in the following sequence TACGGCCG-GENE123-CTCAATCA you would create a primer for the area before GENE123 eg: GCCG, which has the complimentary strand CGGC so you'd make that as your primer.
However as mentioned primers tend to be longer.
It is important to BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi) the sequences to ensure that they won't accidentally prime to the wrong region.
There are companies who can design primers for you, however it is important to make sure that you have the sequence because you need to know where and what you are working with rather than trust a 'black box' (or small white tube!)
PCR involves heating your samples and cooling in cycles. This is generally with a PCR machine which costs from £1000/$1500 although you can make one or use different water baths.
The process is basically: [Denaturing, Annealing, Extension, Stabilisation]x20-30 times
Denaturing: The main concept is to heat the sample to 95C which denatures the DNA and makes the strands separate.
Annealing: This allows the primers to bond to the separated DNA strands.Then the sample is cooled to between 50-60 (depends on the primer and buffer) and held there for about a minute.
Extension: This is when the Enzyme works and starts adding nucleotides from where the primer joined the original strand.
At the end of this cycle your DNA will have amplified the target section only so you have your original + the amplified section = 2.
In the next cycle these 2 strands will both be primed and both will multiply so at the end of the 2nd cycle you will have 4 strands. Another cycle and you will have 8, then 16, then 32 and so on until you have amplified the relevant section hundreds of times.
This creates loads of the section of DNA that you need multiplied so that you can then insert it into bacteria or cells. Though you'd want to run some of it on a gel to check that the amplified fragment is of roughly the right size. If you really really need to double check you can send some of your sample to get sequenced although that can take time and be expensive. Unless your primer designing is totally rubbish it's unlikely that you'd have anything that would be of similar size that isn't your target.
So now that you know the bare basics of how this works, here is a link to the Wikipedia article detailing the other methods as there are now too many to list here without crowding this article up.
Getting something sequenced
Regardless of how they do it various companies offer sequencing for a price.
A fairly complete list can be found at Nucleics.