How to do Plant Genotyping?
Are you interested in DNA and want to do some forensic activities? To give you simple idea (not the detailed and complicated one) on what’s going on in the lab, here’s the overview:
What is genotyping?
From Wikipedia, “genotyping is the process of determining differences in the genetic make-up (genotype) of an individual by examining the individual's DNA sequence using biological assays and comparing it to another individual's sequence or a reference sequence. It reveals the alleles an individual has inherited from their parents. Traditionally genotyping is the use of DNA sequences to define biological populations by use of molecular tools. It does not usually involve defining the genes of an individual”.
In my own words, genotyping is all about digging useful information using DNA samples extracted from plants or humans. That information is necessary to unravel the mystery and solve a particular problem.
"Lab Life" activities:
Plant Genotyping: (rice plant as example)
1. Leaf Tissue Sampling
Whatever study and your purpose might be, you need SAMPLES. Without them, you can’t start in the lab. Two to three leaves from a 21 day old plant (or younger) is enough.
Note: Younger the samples, the better.
2. DNA extraction and quantification
Once you have the samples, extraction of the DNA will be next. There’s a lot of DNA extraction method available out there like CTAB (cetyl trimethylammonium bromide) method. Protocols are easily accessible in the internet. You can even use materials found in the kitchen. Whatever method you want to use, just make sure contamination is completely avoided.
After extraction, checking the quantity and quality of the DNA is very essential to avoid headache later on. You can quantify the DNA using Agarose Gel or by using a spectrophotometer like nanodrop.
3. PCR (Polymerase Chain Reaction)
In PCR, DNA of a particular sequence will be amplified to generate thousands to millions of copies. It’s like cooking or baking, you just need to mix all the ingredients before putting them to the thermal cycler (it's like oven).
PCR components: DNA, buffer, oligonucleotides, primers: F & R, TAQ and water
4. Electrophoresis and Gel Visualization/Documentation
If the PCR is successful, it will produce thousands or millions of copies of DNA, now we need to visualize the bands of those DNA (how they look like as compared with the parents or reference DNA). Electrophoresis and Alpha Imager (special software) will help us come up with the picture of the DNA.
From Wikipedia: “Electrophoresis is a procedure which enables the sorting of molecules based on size and charge. Using an electric field, molecules (such as DNA) can be made to move through a gel made of agar or polyacrylamide. The molecules being sorted are dispensed into a well in the gel material. The gel is placed in an electrophoresis chamber, which is then connected to a power source. When the electric current is applied, the larger molecules move more slowly through the gel while the smaller molecules move faster. The different sized molecules form distinct bands on the gel”.
5. Marker Analysis
Now the most exciting part in my opinion: data interpretation! This will somehow help us answer our problem or the mystery with the help of references available and softwares like GGT, Powermarker, Qgene, Icimapping, TASSEL, Cartographer, R, NTSYS, etc...
a. Score the bands based on your chosen code or the software requirement
b. Format (based from the software requirement) and run the data
c. Interpretation of result. Consult as many related references as possible to make the interpretation easier and credible.
And that's it!
It is very essential to know the theories but without application and hands on experience, you will not appreciate the details that much. By doing, you will discover unexpected things books can’t give. So I can say that learning is incomplete if theories and applications do not meet.
Hope you find this simple article helpful. I might write the detailed discussion of each step in the coming hubs.
Thank you for dropping by!
More by this Author
Fan of Dragon Ball Series? Read to learn the origin of Kamehameha Wave!
sharing great messages...
Planning to go to El Nido and wondering how much?... Okay let's talk about the "Philippine's Last Frontier."