Molecular Biology and Techniques Flashcards
(36 cards)
What is the difference between a nucleoside and a nucleotide? What are the nomenclature differences for each base?
A nucleoside lacks a phosphate group (sugar and base only) whereas a nucleotide has a 3 phosphates + sugar + base (dNTP).
Nucleotides: adenine, guanine, thymine, cytosine
Nucleosides: adenosine, guanosine, thymidine, cytidine
Application PCR: PCR requires nucleotides NOT nucleosides
What are the purines and pyrimidines? What are their structures?
Purines: Adenine Guanine (Pure As Gold)
Pyrimidines: Cytosine, Uracil, Thymine (CUT the PY)
Purines are double ringed; pyrimidines are single ringed
What is Watson-Crick base pairing? What indications does it have on hydrogen bonding?
G-C: 3 hydrogen bonds
A-T: 2 hydrogen bonds
If the phosphate group on a nucleotide is cleaved, what effect does it have on its acidity?
The phosphate group gives the nucleotide its acidity. Therefore, the pH would increase
The first step in DNA replication is separation of the DNA strands. What three proteins/enzymes are required and what are their functions?
- DNA gyrase aka class II topoisomerase: uncoiling DNA AHEAD of fork
- Helicase: unwinding DNA at fork
- Single-strand binding protein (SSB): prevents DNA from adhering to each other once separated - stabilizes single strands
What is the phenomenon associated with the lagging strand and why does it occur? How many primers does Primase lay down on the lagging strand and leading strand?
Okazaki fragments are a result from the complementary antiparallel nature of DNA. Primase is required to lay down multiple primers on the lagging strand in order to keep up with the replication fork, whereas Primase lays down only ONE primer on the leading strand
What is the name of the polymerase that adds nucleotides in the 5’ to 3’ direction?
DNA pol III
Before the Meselson and Stahl experiment, what were the three possibilities about the nature of replication? Based on the experiments, what would they have shown?
- Conservative - only heavy and light
- Semi-conservative - intermediate and light after second round
- Dispersive - intermediate only
What are the functions of DNA Pol I and III?
DNA Pol I - replaces RNA primers with DNA, base excision repair
DNA Pol III - DNA replication
What are the two polymerases responsible for repair DURING replication and what are their modes of action?
DNA Pol III has 3’ to 5’ exonuclease activity whereas DNA Pol I has 5’ to 3’ activity
What are the names of three modes of repairing mutated DNA?
- Base excision repair
- Nucleotide excision repair
- Mismatch repair
What are four different ways that confer base excision repair?
- Deaminated bases
- Alkylated bases
- Oxidized bases
- Accidental uracil
What are the steps of base excision repair?
- Recognition by lesion-specific DNA glycosylases. Damaged base removed leaving AP site
- AP endonuclease cuts out sugar and phosphate (rest of nucleotide)
- Exonuclease removes surrounding nucleotides
- DNA Pol I fills in nucleotides
- DNA ligase
What are three main functions of DNA Pol I
- replaces RNA primers with DNA
- Base excision repair
- Nick translation
How do you determine palindrome sequences on double stranded DNA?
Read the sequence 5’ to 3’ on the top strand. If its the same as you read it from 5’ to 3’ on the bottom strand, then its palindromic
5’GAATTC3’
3’CTTAAG5’
What are the two types of ends that restriction enzymes make when they cut double stranded DNA? Which ends can hybridize?
There are two types of ends - sticky and blunt. Only sticky ends can hybridize
For gene cloning to be successful, what three characteristics must as plasmid possess?
- Restriction site
- origin or replication
- antibiotic-resistant genes
List and describe three steps of PCR
- Denaturation - increase temp to denature DNA
- Annealing: cool reaction to allow hybridization of excess primers
- Elongation: heat-stable polymerases to extend primers
What are the three key features of mRNA codons that allow it to function as the template for translation?
- Continuous: one codon after another without any nucleotides in between
- Non-overlapping: one nucleotide in a codon cannot belong to another codon
- Degenerate: there are multiple codons for a single amino acid
What are two types of mutated codons?
- Missense codon: change in nucleotide as to translate into a new AA
- Nonsense codon: change in nucleotide as to translate into a stop codon
How is translation started and stopped? What is necessary for translation to begin?
Initiation and termination codons begin the peptide chain. However, translation process begins by the formation of the initiation complex. The initiation complex begins to form when the small subunit of the ribosome recognizes the Shine-Dalgarno sequence in the case of prokaryotes or the Kozak sequence in Eukaryotes
Stopped- release factors with GTP bound bind to termination codons
What are the sequences on the initiation and termination codons?
Initiation = AUG Stop = UAA, UAG, UGA
Is there a tRNA for EVERY codon?
No. Stop codons signal for protein “release factors” to bind and dissociate the ribosomal complex and the newly synthesized peptide chain
How are prokaryotic and eukaryotic mRNA differentiated?
Post-transcriptional modification - bacteria do not have 5’ cap and 3’ poly A tail
