Molecular Techniques and Evolution Flashcards
(45 cards)
Briefly describe the steps of recombinant DNA technology.
Vector + DNA fragment —> recombinant DNA –> replication of recombinant DNA within host cells —> isolation, sequencing, and manipulation of purified DNA fragment.
__________ enzymes and DNA ______ allow insertion of DNA fragments into cloning vectors.
Restriction; ligases
What is EcoRI?
A restriction enzyme that makes staggered cuts at the specific 6-bp palindromic sequence GAATTC, yielding fragments with single-stranded complementary 4-base “sticky” ends.
What is ligase’s role in DNA recombination?
Ligase (links fragments during DNA replication) links DNA fragments with either sticky end or blunt ends into vector DNA.
Vector DNA sticky ends base-pair only with the complementary sticky ends on the genomic DNA EcoRI fragment.
(T/F) T4 DNA ligase covalently joins the sugar-phosphate backbones on each strand.
True!
What are plasmids?
Circular, double-stranded DNA molecules that replicate separately from a cell’s chromosomal DNA.
What are the three things that plasmid vectors contain?
1) A synthetic polylinker region containing the only copy of several restriction enzyme recognition sequences, into which an exogenous DNA fragment can be ligated.
2) A selectable gene, such as AMPR, encoding the enzyme B-lactamase, which confers resistance to ampicillin.
3) A replication origin (ORI) sequence where DNA replication can be initiated by host-cell enzymes.
Match the following terms to their definitions regarding DNA cloning in a plasmid vector.
1) Vector preparation
2) Transformation
3) Selection
4) Plasmid regulation
A) Some E.coli cells that are mixed with recombinant vector DNA and subjected to a stress such as heat shock will take up the plasmid DNA.
B) Plasmid DNA replicates and segregates into daughter cells, forming an ampicillin-resistant colony from proliferation of each cell containing the cloned DNA. All colony cells contain plasmids with the same inserted DNA.
C) A DNA fragment is ligated into the POLYLINKER region of a plasmid vector containing an ampr
D) Only the cells containing the plasmid and expressing B-lactamase survive on ampicillin-containing medium
Vector preparation: A DNA fragment is ligated into the POLYLINKER region of a plasmid vector containing an ampr
Transformation: Some E.coli cells that are mixed with recombinant vector DNA and subjected to a stress such as heat shock will take up the plasmid DNA.
Selection: Only the cells containing the plasmid and expressing B-lactamase survive on ampicillin-containing medium
Plasmid regulation: Plasmid DNA replicates and segregates into daughter cells, forming an ampicillin-resistant colony from proliferation of each cell containing the cloned DNA. All colony cells contain plasmids with the same inserted DNA.
How can you screen for successfully ligated plasmids with gene of interest?
Screening involves alpha-complementation.
Bacterial cells are able to make both subunits of beta-galactosidase; from LacZ omega gene and LacZ alpha gene. Beta-galactosidase can digest X-gal and the bacterial cells become blue.
If the insert is ligated with the vector, LacZ alpha gene is disrupted, only lacZ omega gene is made and there is no alpha-complementation. The cells are white.
If the insert is not ligated with the vector, vector is going to be transformed into bacteria where both subunits are made. The cells are blue.
What are the three major steps of PCR? What are its three key components?
Major steps
1) Denaturation: separating target DNA strands
2) Annealing of primers
3) Extension process of replication
*repeat cycle many times to amplify
Key components:
1) Taq DNA polymerase
2) dNTPs
3) DNA template
(T/F) Like in vitro replication, we can also do in vitro transcription.
True!
RNAi (RNA interference) with double-stranded RNA causes an __________ of specific gene function.
Inhibition
*this allows us to study the function of the gene
How is siRNA produced in vitro?
Two plasmid vectors containing the target gene coding sequence in OPPOSITE orientations adjacent to a promoter.
Then, transcription of both vectors occurs using RNA POLYMERASE and ribonucleoside triphosphates. This yields many RNA copies in both orientations, which hybridize to form dsRNA.
This dsRNA is injected into cells and cleaved by DICER into siRNAs.
Why does RNA interference (RNAi) produce siRNAs and not miRNAs in vitro?
The vectors have complementary gene sequence and they hybridize forming 100% complementary base pairs. miRNAs do not have 100% complementary base pairs, but siRNAs do.
What is the mechanism for RNAi to inhibit gene function?
After producing the siRNAs, RISC mediates recognition and hybridization between one strand of the siRNA and its complementary target mRNA sequence.
Then, specific nucleases in the RISC cleave the mRNA-siRNA hybrid.
No gene to translate from; thus no gene function.
What is shRNA? How/why is it made?
Double-stranded small hairpin RNA (shRNA)
Instead of inserting a huge part of gene of interest to make dsRNA, you can insert a hairpin construct, which contains a tandem arrangement of both sense and antisense sequences of the target gene.
This is transcribed to shRNA that is cleaved by Dicer to form siRNA.
It can be used for precise targeting.
What does CRISPR stand for?
What does Cas stand for?
Clustered, Regulatory Interspaced Short Palindromic Repeats.
CRISPR associated proteins
What does the CRISPR-Cas do in bacteria and archaea?
CRISPR-cas constitute an RNA-mediated defense system in bacteria and archaea which protect them against viruses and plasmids.
Match the following steps of the CRISPR-mediated immunity done in bacteria and archaea:
1) Step 1
2) Step 2
3) Step 3
A) CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus, processed, and bind to the Cas protein.
B) The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid and the Cas proteins degrade the nucleic acid.
C) A copy of the invading nucleic acid is integrated into the CRISPR locus.
Step 1: A copy of the invading nucleic acid is integrated into the CRISPR locus.
Step 2: CRISPR RNAs (crRNAs) are transcribed from this CRISPR locus, processed, and bind to the Cas protein.
Step 3: The crRNAs are then incorporated into effector complexes, where the crRNA guides the complex to the invading nucleic acid (even in the future) and the Cas proteins degrade the nucleic acid.
Single nucleotide mutations can be introduced into the genome using an engineered ________________ system.
CRISPR-Cas9
How can you engineer a CRISPR-Cas9 system?
Construct a plasmid encoding Cas9 and another plasmid encoding the guide RNA (guides Cas9 to the target of the gene of interest).
Express these components by transfection with plasmids or by direct injection of Cas9 mRNA and guide RNA.
What are the two components of the guide mRNA?
1) A sequence that folds into a hairpin scaffold structure that binds Cas9.
2) A sequence of 20 nts corresponding to the targeted site in the gene.
After finding the region of interest, what dose Cas9 do (aka its mechanism)?
Base pairing between guide RNA and its complementary genomic DNA sequence directs Cas9 complex to the targeted region of the genome.
Two distinct Cas9 nuclease activities CLEAVE both strands of the target DNA adjacent to the heteroduplex formed with the guide RNA.
What are the two possibilities to repair-target gene inactivation (due to cleavage of both strands by Cas9)?
How do these introduce mutations (which kind)?
1) Nonhomologous end joining (NHEJ): repairs the ds target gene cleavage, but removes a few bases at the cleavage site, inactivating gene function due to FRAMESHIFT MUTATION.
2) Homology-directed repair (HDR): inclusion of a ~100 nt single-stranded DNA segment that spans the sequences flanking the cleavage site along with the Cas9 mRNA and the guide RNA causes homologous recombination repair, which can introduce SINGLE BASE CHANGES in the repaired genomic DNA.
