BioTech Practice
DNA has two strands linked together.
DNA is antiparallel, meaning that it has one side that goes from 3’ to 5’ and the other side is the opposite, 5’ to 3’.
A:T G:C
A purine bases A and G while pyrimidine bases T and C. When A: T it makes 2 bonds while C: G makes 3 bonds.
Phosphate and Sugars.
A nucleotide has a nitrogenous base. The nitrogenous base determines what type it is (ex: A, T, G, C).
The backbone provides the “energy” for the base pairs.
The base pairs are anti-parallel but are replicated using each other.
What is the complementary strand to this strand of DNA: AATTCCGAC
TTAAGGCTG
Meat Tenderizer breaks down the DNA.
Soap pulls apart the membrane and releases DNA.
Ethanol causes the DNA proteins to come out of the solution.
The crushing accelerates DNA diffusion.
DNA Polymerase III: builds the new strand of DNA: reads 3’ to 5’ and builds 5’ to 3’ Topoisomerase: an enzyme that breaks down the backbone to prevent super-coiling. Telomerase: the ends on the DNA to make sure no important code is lost. -It makes repeating, non-coding sequences at the end Primase (RNA): marks down the parts in Okasaki fragments so that DNA polymerase can see it and make nucleotides for it. DNA Polymerase I: adds DNA Nucleotides to points that RNA Primase places down Ligase; “glues” together Okasaki fragments after they’re “cut.” What does 3’ to 5’ mean? Why is it important? 3’ to 5’ is the direction of the DNA “molecules” depending on their carbon structure. Why are the two strands of DNA called antiparallel? They are anti-parallel since they are “parallel” but are opposite of one another. That means that one side is 3’ to 5’ and the other side is 5’ to 3’. What is the difference between the leading strand and the lagging strand? The leading strand is the “easier” to make strand since it does not need to be cut up into fragments. The lagging strand is the strand that needs to be cut up into fragments where DNA Polymerase I would “see” the RNA Primer and replaces it with DNA nucleotides. Describe an Okasaki fragment and how it is created. Why is the lagging strand built in fragments since that seems inefficient and slower? An Okasaki fragment are fragments that are being made of lagging strandsProteins: Proteins are made up of long chains of amino acids.Proteins vary in both sequence and number of amino acids The primary structure of a protein is its specific amino acid sequence. What causes the protein to fold into its final 3D shape? Multiple chains will interact with each other which causes the protein to fold into its final 3D shape. The amino acid sequence itself “codes” for the way the protein should fold. This also determines the functionality. In humans, there is Insulin and Histone as proteins. In other organisms (specifically plants), there are glutelins and lectins.
‘Enzymes are proteins linked together.
Enzymes are important for reactions in the body and have many functions.
(Example: Ligase)
What is the enzyme/protein called that reads a RNA strand and builds a chain of amino acids?
The enzyme that reads an RNA strand and builds a chain of amino acids is the ribosome.
Aminoacyl tRNA synthetase bonds amino acids to tRNA
tRNA “transfers” the amino acids from the anti-codon.
mRNA codes for proteins.
tRNA and mRNA work together.
rRNA also helps make the proteins/chain of amino acids.
RNA Polymerase: rRNA, mRNA, and tRNA.
Central dogma:
mRNA is similar to DNA but it only has one strand, uses U instead of T, and is missing H3c on it’s 2’ Carbon
How much DNA is read and copied into mRNA at a time? In other words, how big is the transcription bubble and what enzyme does transcription?
In prokaryotic cells, it copies about 300-500 bases in the gene.
-20 bases at a time.
In Eukaryotes ONLY, DNA cannot leave the nucleus.
RNA Polymerase does this and builds RNA like DNA Polymerase III (5’ to 3’).
What is the difference between the transcription bubble and the replication bubble?
The replication bubble replicates the entirety of the DNA while the transcription bubble only makes the RNA for the specific part of the DNA it needs to make a specific protein.
Why is it necessary for a copy of a gene to be made (in the form of mRNA)?So that the RNA does not code for anything else that is not needed, it is necessary for a copy of a gene to be made in the form of mRNA. Also, for accuracy purposes, a copy of a gene is made so that if there are any errors, the “next” set would not have those same errors.
What is the difference between introns and exons?
Introns: the parts of the RNA that are “turned off”Exons: the parts of the RNA that are “turned on” or are being used
-Both are still needed to make a specific protein since if it was only exons, it would code for a different protein.
Where does transcription occur in a cell? Where does translation occur? Answer for both Prokaryotes and Eukaryotes.
Prokaryotes: Transcription and translation occur in the cytoplasm.
Eukaryotes: While transcription occurs in the Nucleus, and translation occurs in the cytoplasm.
What is the difference between prokaryotic and eukaryotic cells?
Prokaryotic: No nucleus, no introns no exons only a cytoplasm, simple proof-reading, transcription occurs in cytoplasm, etc.
Eukaryotic: has a nucleus, has introns and exons, has the DNA in the nucleus and is not allowed to leave it, advanced proof-reading, etc.
Describe the three main types of RNA as well as the enzyme that builds each different type.
RNA Polymerase I: rRNA (ribosomal RNA ) : makes ribosomes which are used during translation.
RNA Polymerase II: mRNA ( Messenger RNA): (the genetic code that gives instructions on how to make proteins. )
RNA Polymerase III: tRNA (transfer RNA): makes amino acids
- Summarize the central dogma as the following steps: Include the names of the processes between the steps and explain what each process does briefly in the space below.
DNA (gene) → RNA → Protein
DNA -> RNA using transcription which occurs in the nucleus for eukaryotes and occurs in the cytoplasm for prokaryotes.
Transcription takes about 300-500 base pairs of DNA (Only a part of it) and basically makes the RNA code.
-From DNA Nucleic Acid language to RNA Nucleic Acid Language
RNA - Protein
b. Translation which occurs in the cytoplasm for both eukaryotes and prokaryotes. RNA Polymerase (different types( makes the specific type of RNA.
-From Nucleic Acid language to Amino Acid language
Intracellular and extracellular proteins are made in different locations. What are these locations and why can’t they be made in either place?
Since they have different functions, are “packaged” differently and have some form of different structures they cannot be put in the other place. For example, a membrane bound extracellular protein cannot be used for intracellular functions.
Restriction enzymes. What are they? Why do bacteria have them? Why do humans use them?
Restriction enzymes cut DNA into smaller pieces.
Bacteria uses restriction enzymes to defend themselves against “bacterial viruses”.
Humans have them to also protect us “unknown DNA”.
What is a plasmid and why are they important?
Plasmid is “tiny” DNA found in bacteria, they are:
-used to transfer genetic information in bacteria
-the reason why they evolve fast
-used in a lot of labs and are useful for research,
Transcribe and translate the following template of DNA. You will need to look up an amino acid chart. You should name and explain the use of the proper enzymes and molecules for transcription and translation. Assume this is the beginning of the DNA, so don’t start translating until you hit the ‘start codon’!
3’ A C G C G C A T G T A C C G A G C A C G G T A G A T T C G C…5’
5’ U G C G C G U A C A U G G C U C G U G C C A U C U A A G C G….3’
1: AUG (Start)
2: GCU (Ala)
3: CGU (Arg)
4: GCC (Ala)
5: Auc (Ile)
6: UAA (Stop)
Think about all of the experiments we have run throughout this year so far and list them. Can you explain how to do each one? Would you be able to show someone else how to do the experiment? Why do we do the experiments? In other words, what do the results tell us about our test subjects?
-In the “Personal DNA Extraction” experiment, you put distilled water in your mouth and spit it out after about a minute and add it to a test tube. Add a little bit of water and add a drop of detergent to the test tube. Then, you would add a piece of meat tenderizer to the test tube and let it sit for about 10 minutes. Finally, using a pipette slowly add alcohol without mixing it and let it rest for 2 minutes. Using a pipette, suck out the DNA and place it into a microcentrifuge tube. The results tell us about how we were able to make DNA “visible” by using things like a meat tenderizer.
-In the “Making Solutions” experiment, we essentially made the solutions for a lot of the future experiments we made. We first had to calculate what we needed to make (the amount of powder, etc.) and made solutions using graduated cylinders and different types of buffers. We did this experiment so that in future experiments, we would not need to make each one at a different time which would take a lot more time than if we made them all at once and stored them for use at a later time.
-In the “Casting Agarose Gels” experiment, we first measured the agarose powder required and did all the proper steps to make the gel that we would use to try placing “samples” using a micropipette. We did this experiment to gain practice in being able to accurately use the micropipette.
-In the “Making Microbiology Media” experiment, we made the specific solutions that we need to make that will be needed for the future lab which is to grow bacteria! We did this experiment to see how we are able to grow bacteria “from the very beginning”.
-During the aseptic technique lab, we practiced growing only a small part of the bacteria that we wanted and also did the proper procedures to kill all the bacteria around us that we did not want, making a “sterilized bubble”. We used alcohol lamps and sterilized the loop using the lamp to make sure there were no unwanted living bacteria that would grow as well. We did this experiment so that in many labs that need sterilization, we had practice with it. There were multiple different results with some people only growing the bacteria they wanted while others grew other types of bacteria which showed the importance of sterilizing the materials properly before conducting the experiment.
-In the microbes and health lab, we added milk to the right side and yogurt to the left side of the plate and straked each plate using the plate technique which we practiced in the previous lab. This lab gave more practice ==on plating the plates properly since it needs a lot of accuracy.
-In the gram staining lab, we used all of the practice on the previous alb and grew e-coli, yogurt, and milk by using the aseptic technique and streaking the plate! Later, we were able to see the results of the bacteria we grew and how well it grew depending on how we streaked the plate. The lab gave us a lot more practice with plating bacteria but also showed how when we properly used the specific techniques, we were able to grow the bacteria we wanted without any issues.