Deoxyribonucleic acid, abbreviated as DNA, encodes all the traits that any organism possesses, and certain combinations of DNA nucleotides create different genotypes or pairs of traits. The characteristics represented in a genotype can be dominant or recessive and will define the way in which the organism expresses that trait. To determine a genotype, you can use a Punnett square, but if you work in a more advanced laboratory, you can use analytical methods such as PCR analysis and nucleic acid hybridization to check which genotypes are present.
Steps
Method 1 of 3: Work with Punnett square
Step 1. Draw a 2 x 2 grid
The Punnett square is used to determine the probability of the genotype of the offspring from the genotypes of their parents. In addition, it must be labeled with the genotype of each parent and, inside, the possible genotypes of the children must be shown.
Step 2. Label the left side
Take the genotype of one of the parents and divide the two letters (which represent dominant and recessive traits). Put one letter to the left of the top row and the other to the left of the bottom row. In this way, the father's contribution to the son's genotype will be represented.
It is common to put a dominant trait (capital letter) in the top row and a recessive trait (lowercase letter) in the lower part if the two traits are different
Step 3. Label the top
The traits of the other parent should be divided in the same way, but this time place one letter on top of the left column and the other on the right column. This will represent the contribution of the second parent to the child's genotype.
It is common to put a dominant trait (capital letter) on the left and a recessive trait (small letter) on the right, if the two traits are different
Step 4. Enter the known information
Now you can fill in each square and, inside each one, write the letter that corresponds to the row in which the square is located. Next, write the letter that corresponds to the column in which the box is located. This will identify all the possible genotypes for the children as well as the percentage in which they would occur.
For mixed traits, write the genotype so that the dominant trait appears first (Rr instead of rR)
Method 2 of 3: Sequencing with PCR
Step 1. Select a primer
A primer is a molecule that binds to a particular DNA sequence. Once bound, the binder can be detected to determine whether or not the sequence was present in the sample. This allows testing of specific sequences that correspond to a particular genotype.
Step 2. Collect a DNA sample
When you have selected a primer that binds to the sequence in question, you will need to extract DNA from the cell. Follow the appropriate extraction protocol for your laboratory. When you have collected a sample, you can examine it.
Step 3. Add the primer to the sample
Add the primer to the DNA sample. If the sequence corresponding to this primer is present, it will bind to the molecule, and when the process is complete, you can proceed to analysis.
Step 4. Analyze the results
In simple cases, the results merely reappear as positive or negative based on whether or not the primer was bound to the DNA strands. Some more complex methods may require post-PCR procedures. These are techniques that can be time consuming and expensive, so they should be avoided as much as possible.
Method 3 of 3: Use Nucleic Acid Hybridization
Step 1. Digest a DNA sample
DNA digestion is a process that breaks down the two chains of deoxyribonucleic acid. In this way, each strand is left without its complementary base pair and this opening allows DNA to join other strands of that type.
Step 2. Separate fragments by electrophoresis
Electrophoresis is a process that uses electrical current to move molecules through a gel. In this case, you must use agarose gel. The DNA will reach the positive end of the gel and will separate according to size.
Step 3. Transfer onto nylon or nitrocellulose paper
When the chains have separated, they should be transferred out of the gel. Use a Southern blot procedure to transfer the sample to a sheet of nylon or nitrocellulose paper. These are more appropriate means of adding the probe.
Step 4. Add a probe
Probes are strands of DNA that complement the strands in question. If the fragment representing a specific genotype is present, it will bind to the probe. It also contains a fluorescent molecule that will be detected during the analysis.
Step 5. Wash the paper
After allowing enough time for the probe to bond with any samples present, you will need to wash the paper. Follow your specific lab procedures, although generally only the paper needs to be lightly rinsed with water. Be careful not to contaminate or spoil the sample during washing.
Step 6. Expose the paper
When the sample has been washed, you can examine it. By exposing the sample to ultraviolet light, the fluorophore attached to the probe will be excited, producing an image with areas of intense light relative to the background. If no probe is present, no illuminated areas will appear.