Lecture 8 Flashcards
What does Recombinant DNA technology allow for?
Allows for scientists to identify, isolate and combine DNA fragments from any organism to obtain the desired properties in new DNA molecule that can be maintained in bacterial
What does Polymerase chain reaction (PCR) allow for?
Allows for the amplification of small to medium-sized DNA fragments from minute amounts. RNA can be amplified after copying it into DNA
What does DNA sequencing allow for?
Allows the determination of the nucleotide sequence of a DNA fragment (and RNA after copying it into DNA).
What does CRISPR/Cas genome editing all for?
Allows for the precise manipulation of DNA and in the case of Cas13, RNA, in a wide range of organisms. The Cas9 enzyme cuts DNA in a precise location because it is guided to the the target sequence through the presence of a “single guide RNA”
What can CRISPR be used to generate?
Simple mutations or complex gene editing (gene replacement) approaches
What is a mutation?
Change the DNA sequence of a given gene to create a mutant organisms. This includes classic (random) and modern (targeted) mutations
What is Transgenesis?
Introduce a new piece of DNA to the genome to create a transgenic organism
What does Transgenesis result in?
Because a new piece of DNA is introduced this causes the organism to produce a new type of protein that has some new property
What is Gene editing?
A fairly new technique (CRISPR/CAS9) is a blend of mutations and transgenesis
What are the 2 ways you can clone a gene?
Using PCR or retrieve it from a DNA library
What can DNA Libraries be used for?
Genomic DNA or cDNAs
What is the idea behind DNA libraries?
The idea is to have an entire genome or transcriptome represented in individual plasmid DNAs (vector DNAs)
How are different fragments of DNA created in DNA Libraries?
Genomic DNA is digested by restriction enzymes to create different fragments
How do Restriction enzymes know where to cut?
They recognize specific DNA sequences
What occurs in Reverse Transcriptase?
An RNA template is used to make DNA
Who invented PCR?
Mullis
Why are Thermocyclers necessary in PCR?
It is needed to tear the double stranded DNA apart
How do we clone our GOI into a suitable vector?
Restriction enzymes recognize specific DNA sequences and cuts it to create sticky ends (ends with overhangs). Then the vector DNA and PCR fragment are cut to make the DNA ends compatible and ligase is added to fuse them together
What are sticky ends?
When DNA is cut by restriction enzymes they create 5’ overhangs called sticky ends
When is DNA said to be clones?
Upon successful transformation of the recombinant DNA into the bacteria
What does Transposase do?
Integrates the transposon DNA into new integration sites
How is Transposase created?
A helper plasmid encodes a function copy of Transposase
What does the P-Element contain?
The vector with the recombinant DNA that contains recognition sites for the Transposase
How is Recombinant DNA added to a fly genome?
Both the P-element vector and the helper plasmid are co-injected into the fly embryo which causes insertion of the P-element into the host genome
How can we know if integration of a gene into a drosophila is successful?
Successful integration can be monitored by expression of the w+ gene (makes red eyes) the hose genome is w- so it has white eyes
How does Sanger Sequencing work?
A DNA template and normal nucleotides (dNTPs) undergo synthesis by DNA polymerase. In each tube there is a certain nucleotide type of (ddNTPs) in small amounts that will block the activity of DNA polymerase creating different fragments
What are the normal nucleotides in Sanger sequencing?
dNTPs
What are the abnormal nucleotides in Sanger Sequencing?
ddNTPs that are missing both OH groups
What occurs after replication in Sanger Sequencing?
Since the fragments also contain radioactively labelled dNTP we can see their length. The fragments then become denatured and the single strands are separated by gel-electrophoresis
What is different about Automated Sanger Sequencing?
The ddNTPs are labelled with fluorescent dye and after replication and electrophoresis the fluorescent dyes are detected using a detector. This all takes place in a single reaction tube rather than 4.
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
Where was CRISPR discovered?
In Bacteria and Archaebacteria
What does CRISPR do in Bacteria and Archaebacteria?
Serves as memory against phages
How does CRISPR help Bacteria and Archaebacteria?
DNA from an invading phage is stored in a specific locus if the bacteria chromosome. Should another infection occur from a phage with the same DNA the stored CRISPR DNA is used to identify the invading DNA and cut it via Cas nucleases
What are Cas nucleases?
DNA degrading enzymes that are guided to their target by CRISPR-derived RNA
Why are Cas nucleases known as engineered nucleases?
Because the enzyme is recruited to specific locus
What would the Cas nuclease result in?
A double strand break
What is the simplest way that CRISPR allows the double stranded break to be repaired?
Non-homologous end joining
What will NHEJ cause?
Small deletions in the targeted region resulting in gene mutation
Why is NHEJ not gene editing?
Because we don’t technically have control on what the final sequence will be
What did Homologous recombination depend on?
The sister chromatid to fix the double stranded break
What is it called when CRISPR depends on homologous recombination?
Homology-Directed Repair (HDR)
What occurs in advanced CRISPR?
a donor DNA that is homologous but not identical to the region with the double stranded break is injected. The fools the HR system to use the injected donor
What is Cas9?
An RNA-binding nuclease
What is the single RNA that is incorporated into the Cas9 enzymes function?
It serves as a guide to direct the CAS9/RNA complex to DNA that is complimentary to the RNA so that it can introduce a double stranded break
Why is the RNA in the Cas9 enzyme called the single guide RNA?
Because it guides the complex to the complementary DNA
What is the PAM sequences?
The genetic sequence adjacent to the RNA-DNA match that needs to present next to the target sequence for initial binding of the Cas9 effector complex
