Chapter 20: DNA Tools and Biotechnology Flashcards
What is the Sanger method (Dideoxy sequencing) used for?
Isolating a specific DNA sequence
Explain the steps of Sanger method (Dideoxy sequencing).
Step 1 - Separate DNA strands to have a single template strand Step 2 - RNA primer and DNA polymerase are added - Template strand is evenly distributed into 4 test tubes that have different fluorescent deoxyribonucleotides Step 3 - DNA strands of different lengths are synthesized - The glowing tags are designed to make the strands stop replicating and fall off once the specific tag is reached (ATCG) - This is done many times in order to figure out what the entire sequence looks like
Below is a spectrogram generated by dideoxy chain termination method of DNA sequencing (Sanger sequencing). What is the sequence of the original DNA template? GAAATTGTTATCCGC (Short) (Long) A. 5’ - GAAATTGTTATCCGC - 3’ B. 5’ - CTTTAACAATAGGCG - 3’ C. 5’ - CGCCTATTGTTAAAG - 3’ D. 5’ - GCGGATAACAATTTC - 3’
D The shortest labeled strand is the 5’ end, therefore, when we replicate the given sequence, we must replicate it from 5’ to 3’, requiring us to read it backwards.
The shortest labeled strand is the ____ end.
5’
The longest labeled strand is the ____ end.
3’
What is DNA cloning?
An experimental method used to create multiple copies of a specific DNA sequence
What is Polymerase Chain Reaction (PCR) used for?
Making multiple copies of the target gene In 3 hours you could make 1,000,000 copies
What are the 3 steps of Polymerase Chain Reaction (PCR)?
- Denaturation - Sample is heated to 95° to separate the DNA strands 2. Annealing - RNA primers, fluorescent nucleotides and Taq polymerase are added - At 55° to attach primers 3. Extension - DNA strand is replicated - At 72° to help the strand be made
What would be the outcome of PCR reaction in which primers were used that were complementary to more than one DNA sequence?
The DNA target sequence would be amplified, but to less then the expected number of copies.
If you want to make enough copies of insulin for a patient, PCR would be inefficient. Explain why and explain what other method you could use to replicate insulin fast.
PCR would be inefficient because you do not have time to wait 3 hours while your patient needs insulin. Instead, you would use bacteria.
Why is bacteria a very good DNA amplification factory?
- Already contain all necessary components for DNA replication (Nucleotide bases, enzymes) - Bacterial cells grow rapidly, meaning constant DNA amplification
What is the best way to introduce bacterial cells into the target DNA sequence?
Through plasmids
What is a plasmid?
Circular extrachromosomal DNA that carries genes that are not absolutely necessary for survival
Explain how we can use the plasmid of a bacterial cell in order to replicate the DNA sequence of interest.
- Take the bacterial plasmid out of the bacterial cell 2. Remove a section of nucleotides from the plasmid and insert the gene of interest (Recombinant Plasmid) 3. Place the Recombinant plasmid back into the bacterial cell 4. The bacterial cell replicates the gene of interest 5. The cloned DNA can now be used for whatever you wanted it for
How do you construct a recombinant DNA plasmid?
- The circular plasmid is cut at a specific sequence by a restriction enzyme (creating sticky ends) 2. The target DNA is also cut with the same restriction enzyme in order to match the sticky ends of the plasmid 3. The target DNA is inserted into the plasmid and DNA ligase joins the phosphodiester bonds 4. Now we have the recombinant plasmid
