Molecular Tools Flashcards
Describe the complication with DNA replication associated with the fact that DNA polymerase can only add nucleotides in the 5’ to 3’ direction.
- Lagging strand has to be copied in a discontinuous manner
- Leading strand can be copied in a continuous manner
- Lagging strand has a series of short segments called Okazaki fragments that must be joined together
What is needed to initiate synthesis of a new DNA strand?
- An RNA primer (7 to 10 nucleotides) is needed to initiate synthesis of a new DNA strand
- Synthesized by an enzyme called primase
What are DNA polymerases?
Enzymes that synthesize new polynucleotides complementary to an existing DNA or RNA template.
- DNA-dependent DNA polymerase
- RNA-dependent DNA polymerase
Describe the biological activities of DNA polymerases.
- In the cell, DNA polymerases are essential for DNA replication
- In addition to adding nucleotides, they have enzymatic activities that are important for removing incorrect nucleotides
- 3’ to 5’ exonuclease activity removes incorrect nucleotides (Called ‘proofreading’ activity)
- 5’ to 3’ exonuclease activity is needed for DNA polymerases that must remove part of a polynucleotide that is already attached to the template strand that the polymerase is copying
- For example, in DNA replication, an RNA primer is used to start DNA replication but must be removed by DNA polymerase 5’ to 3’ exonuclease activity and replaced by DNA nucleotides so that newly replicated strands can be ligated together
What are DNA polymerases used for in Molecular Biology research?
What is DNA polymerase I used for in molecular biology research?
DNA labelling
Has both 3’ to 5’ and 5’ to 3’ exonuclease activities
Unmodified E. coli enzyme.
What is Klenow polymerase used for in molecular biology research?
5’ to 3’ exonuclease activity removed but the 5’ to 3’ polymerase and 3’ to 5’ exonuclease activity remains
Modified version of E. coli DNA polymerase I.
The removal of the 5’ to 3’ exonuclease activity from Klenow polymerase is useful because it prevents DNA degradation during key processes like DNA labeling, blunt-end synthesis, and high-fidelity DNA replication, while retaining the necessary 3’ to 5’ proofreading function to maintain accuracy.
What is Taq polymerase used for in molecular biology research?
- PCR
- Isolated from a bacteria (Thermus aquaticus) that lives in hot springs
Thermus aquaticus DNA polymerase I - useful in PCR because it remains active at high temperatures.
Describe the polymerase chain reaction process.
- Denaturation (95C)
- Primer annealing (50-60C)
- Elongation by Taq polymerase I (72C)
What are applications of PCR in food science? [4]
- Detection of foodborne pathogens (multiplex PCR to detect for multiple foodborne pathogens)
- Testing for the presence of GMOs
- Making recombinant proteins (e.g., insulin production; chymosin production)
- Identifying and characterizing microbial populations in food products
What is reverse transcriptase used for in molecular biology research?
- cDNA synthesis
- Makes DNA copies from RNA templates
RNA-dependent DNA polymerase, obtained from various retroviruses. Retroviruses (e.g., HIV) use RT to replicate their genomes.
What is reverse transcriptase useful for in molecular biology?
Transcriptomics - analysis of total gene expression of an organism, tissue or cell
Describe a schematic representation of a transcriptomic evaluation approach.
- Tissue sample
- RNA extraction
- Library preparation
- High-throughput sequencing
- Transcriptome assembly
- Differential expression and functional analyses
Describe how reverse transcriptase is used in molecular biology. [7]
- Eukaryotic mRNAs have a poly(A) tail at their 3’end
- Add a primer (oligonucleotide) with ~20 nucleotides of Ts to bind to the poly A tail
- Reverse transcriptase enzyme synthesizes DNA from the RNA strand
- Add Ribonuclease H to degrade most of the RNA strand
- Synthesize the next strand with DNA polymerase using leftover RNA as primers
- DNA polymerase that still has a 5’ to 3’ exonuclease activity to remove RNA and replace with DNA
- Now you have a double stranded complementary DNA (cDNA) that can be used for DNA sequencing
cDNA has always been made from an RNA template.
What is a nuclease?
Enzymes that degrade DNA molecules by breaking the phosphodiester bonds that link one nucleotide to another nucleotide
What are restriction endonucleases?
- Cut double stranded DNA at a specific recognition sequence
- Called ‘restriction endonucleases’ because in bacterial cells they cut DNA from bacteriophage
Example: EcoRI
What is EcoRI?
- A restriction enzyme that binds DNA as a dimer and cuts both strands
Describe DNA digestion with EcoRI.
Sticky ends, 5’ overhang
Restriction enzymes produce […]
Either blunt or sticky ends
How many restriction endonucleases are available for use in the lab?
- > 600!
- Many have hexanucleotide (6bp) recognition sequences
- Some have degenerate recognition sequences such as Bgl I (some specificity, but many nucleotides can just be whatever)
How can restriction digests be analyzed?
DNA Gel electrophoresis
- Agarose gel - pores ranging from 100 to 300 nm in diameter
- Agarose is a linear polymer extracted from red algae
- DNA is negatively charged and moves towards positive electrode (anode)
- Smaller DNA fragments migrate faster than larger DNA fragments through the gel
How can DNA be visualized after gel electrophoresis?
- Ethidium bromide wash
- DNA migration pattern depends on agarose %
DNA migration pattern depends on agarose %. What % would you use for larger vs smaller fragments?
- to look at larger DNA fragments run 0.75% gel
- to look at smaller DNA fragments run 1.25% gel
Describe the role of DNA ligase in vivo.
- Needed during DNA replication after RNA primers have been replaced by DNA
Desribe DNA ligase in vitro.
- Easier to ligate sticky ends
Give an outline of gene cloning.
- Need a plasmid - circular DNA fragment
- Animal gene has BamHI restriction sites on either side
- After ligation of animal gene into plasmid, transform ligation mixture into E. coli
- Select E. coli that have the plasmid and original E. coli plasmid
Describe pUC8.
- A simple E. coli plasmid
- Used for recombinant plasmid selection
Describe recombinant plasmid selection.
- Done with plasmid pUC8
Describe the structure of the yeast chymosin expression plasmid.
Note from Dr. Measday:
“The chymosin expression plasmid and the pUC8 plasmid are two separate plasmids. I did not show the chymosin expression plasmid before cloning in the chymosin gene but it would have been a plasmid just carrying the ori, two micron, AMP and LEU2 gene with a multiple cloning site. The PGK1 promoter, Pro-chymosin gene and PGK terminator would all have to be cloned into the plasmid. The cloning would all be done in E. coli - this could be done by PCR amplification of the promoter, chymosin gene and PGK terminator. Once the chymosin expression plasmid is created, then it would be transformed into yeast for expression of the chymosin gene.”
What is pPGK1?
- Promoter region from the yeast PGK1 gene (3-phosphoglycerate kinase) - strong, constitutive promoter
What is pPGK1term?
- Terminator region from the yeast PGK1 gene - needed for efficient transcription termination
What is pro-chymosin?
Bovine chymosin gene
What is ori?
Bacterial origin of replication
What is 2µ?
Yeast replication gene that allows for high copy numbers of the plasmid in yeast.
What is LEU2?
- Selection for plasmid in yeast.
- Must transform plasmid into a leu2D strain and select on -leucine agar plates
- Plasmid transformation into yeast can be selected for on agar plates that do not have leucine
What is AMP^R?
- Gene for ampicillin resistance - codes for beta-lactamase
- Must select on ampicillin agar plates
Describe a milk clotting assay with yeast expressed pro-chymosin.
- Pre – signal peptide (16 amino acids, that is lost during secretion - not in yeast plasmid)
- Pro-chymosin – inactive form of chymosin
- At low pH, an autocatalytic cleavage removes 42 amino acids from the N-terminus of prochymosin to form Chymosin which is active
What happens to mRNAs after translation?
Once mRNAs enter the cytoplasm, they are either (1) translated, (2) stored for later translation or (3) degraded.
- They may be temporarily translationally repressed
- mRNAs are actually associated with proteins during their lifetime which are called mRNA-protein (mRNP) complexes
- P-bodies are cytoplasmic mRNP granules that have translationally repressed mRNAs and proteins involved in mRNA decay
Are amino acids the same between all organisms?
- Yes - there are 20 proteinogenic amino acids in all organisms
What is selenocysteine?
- Analogue of cysteine with selenium in place of sulfer
- Not coded for directly in the genetic code
- Not universal in all organisms
- UGA codon (stop codon) can encode for selenocysteine if cells are grown in selenium
- Requires presence of selenocysteine insertion sequence (SECIS) in mRNA
Define non-proteinogenic amino acids and give 2 examples.
- Not incorporated into proteins
- Gamma-aminobutyric acid (GABA) is produced by decarboxylation of glutamic acid
- GABA is found in the human brain where it acts as an inhibitory neurotransmitter
- L-3,4-dihydroxyphenylalanine (L-DOPA) is synthesized from the amino acid tyrosine
- Found in plants and humans
- L-DOPA is the precursor to neurotransmitters including dopamine
- Gamma-aminobutyric acid (GABA) is produced by decarboxylation of glutamic acid
What are nucleases?
Enzymes which degrade DNA molecules by breaking down phosphodiester bonds that link one nucleotide to another; two general categories (endo and exo)
- Endonuclease make internal cuts
- Exonuclease remove from ends
What is a restriction endonuclease?
- Cut double stranded DNA at a specific recognition sequence
What would happen if you cut the lacZ gene in the pUC8 plasmid with two enzymes?
- A fragment would be lost/degraded in the cloning step
- You could then clone in a gene with an EcoRI site on the left side and a BamHI site on the right side
What is the cellular function of beta-galactosidase?
- To split lactose into glucose and galactose
The lacZ’ gene in the pUC8 plasmid is functional and so bacteria containing pUC8 will be blue!
How are recombinant plasmids selected with pUC8?
- When the DNA insert (gene) is cloned into the lacZ’ gene, it disrupts lacZ’ function and therefore X-gal is not cleaved and the bacterial colony will be white.
