DNA Laboratory Methods Part 2

Question 1

Lambda red system

Answer 1

Widely used to create knockouts in bacteria, induces site-specific recombination. Utilizes lambda recombinase/integrase

Question 2

Conservative site-specific recombination

Answer 2

The breakage and joining of DNA occur at 2 special sites- 1 on each participating DNA molecule. The sites flank the gene to be introduced and the gene to be removed. They contain recognition sites for the lambda recombinase that catalyzes the reaction. Utilized by the lambda red system

Question 3

Steps of the lambda red system

Answer 3

1. Bacteria transformed lambda red plasmid containing the antibiotic resistance gene and the gene for lambda red recombinase. Expression is controlled by the inducible promoter
2. Lambda recombinase is expressed and swaps the 2 genes for one another
3. The target gene is replaced with the antibiotic resistance gene

Question 4

attP

Answer 4

One of the specialized sequences flanking the antibiotic resistance gene in the lambda red system

Question 5

attB

Answer 5

One of the specialized sequences flanking the gene to be knocked out in the lambda red system.

Question 6

Lambda integrase

Answer 6

Attaches to the specialized B and P sequences. Mediates the removal of the gene to be knocked out and the introduction of the antibiotic resistance gene.

Question 7

Helper plasmid

Answer 7

The lambda red system may use an additional plasmid (a helper plasmid) that expresses FLP recombinase under an inducible promoter. Removes the antibiotic resistance gene via FLP recognition targets (FRTs)

Question 8

Goal of the lambda red system

Answer 8

To cure the bacteria of lambda and FLP plasmids. They are temperature-sensitive replicons- growing at a certain temperature kicks the plasmids out

Question 9

FLP recombinase recognition targets (FRTs)

Answer 9

Flanks the antibiotic resistance gene, in the situation where a second plasmid is used in the lambda red system

Question 10

FLP recombinase

Answer 10

Recognizes the FLP recombinase recognition targets and removes the antibiotic resistance gene

Question 11

Methods of screening and confirming knockouts (3)

Answer 11

These methods confirm that the knockout actually worked
1. Plate on antibiotic-containing media- only those organisms that have the resistance cassette for the specific antibiotic will grow
2. PCR
3. Sequencing

Question 12

Use of PCR in confirming knockouts

Answer 12

Can confirm that the antibiotic resistance plasmid is there, or can confirm the target gene is missing

Question 13

Sequencing method of confirming knockouts

Answer 13

Used to confirm that the recombination event did not have any pleiotropic effects on the chromosome or plasmid region. Does not shift the open reading frame or mutate neighboring genes

Question 14

Transcription activator-like effector nuclease (TALEN)

Answer 14

A nuclease that is engineered to cleave specific DNA sequences. It consists of a TAL effector DNA-binding domain fused to a DNA cleavage domain

Question 15

TALEN DNA binding domain

Answer 15

Contains repeated, conserved 33034 amino acid sequences with divergent 12th and 13th residues (they are changed). The amino acids are changed because that is how the targeting for the TALEN nuclease is manufactured. By varying the amino acids, it can bind to different sequences in the DNA. Has a repeat variable di-residue (RVD) that shows a strong correlation with nucleotide recognition. TALEN binds to the target gene and uses its nuclease activity to make a double stranded cut

Question 16

Steps involved in TALEN (4)

Answer 16

1. Cells are transfected with the gene for TALEN
2. TALEN binds to the target gene due to its engineered varying amino acids
3. TALEN is expressed and induces a double stranded cut in the gene. This results in either non-homologous end joining (knocking the gene out) or homologous recombination (which can also knock the gene out)
4. Target gene loses nucleotides due to non-homologous end joining. The gene is mutated and the protein is not expressed, technically making it a knockout. However, you can also completely remove the target gene

Question 17

TALEN steps to completely remove and knockout a gene (4)

Answer 17

1. Cells transfected with a plasmid containing antibiotic resistance gene flanked by the same homologous sequences- the sequences should be homologous to the gene you want to knock out
2. Target gene flanked by homologous sequences
3. Homologous recombination occurs to swap the genes
4. Target gene is replaced by an antibiotic resistance gene, and the target gene is knocked out. The protein is not expressed

Question 18

Zinc finger nucleases

Answer 18

Similar to TALEN. Composed of a zinc finger fused to a nuclease. It contains between 3 and 6 individual zinc finger repeats, which each recognize between 9 and 18 base pairs. They bind sequences and cut- NHEJ or homologous recombination

Question 19

Limiting factor in creating knockouts

Answer 19

Engineering the specificity to your gene of interest. We have to change amino acids in the protein to engineer its specificity, as seen with Zinc finger nucleases and TALEN.

Question 20

Benefits of CRISPR-Cas9

Answer 20

Just as specific as other knockout techniques, and it is much easier to engineer specificity. Discovered 12 years ago

Question 21

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Type II system

Answer 21

A naturally occurring system in bacteria that combats bacteriophage infection. When a phage infects bacteria, a piece of the phage DNA is integrated into the crispr locus. Acts like a “library” of prior viral infections. If the same bacteriophage tries to infect at a later time, the crispr locus will be transcribed and will recognize the phage DNA and destroy it, preventing further infection

Question 22

How does the CRISPR-Cas 9 system work? (4 steps)

Answer 22

1. A piece of phage DNA is integrated into the CRISPR locus when the bacteria is infected
2. The crispr locus is transcribed. Small guide RNA is formed, and transcripts for Cas nuclease are formed
3. Cas is translated, and sgRNA is assembled onto Cas
4. sgRNA guides Cas nuclease to complementary phage DNA. The phage DNA is cut. This is a double stranded cut- Cas9 has RuvC and HNH nuclease domains

Question 23

Making knockouts with CRISPR-Cas9

Answer 23

You can engineer a crispr locus on a plasmid to contain the DNA of a target gene which you want to knock out. Then you transfect it into the cell. Once expressed, Cas9 will have sgRNA specific to your target gene. Cas9-sgRNA will then cut the gene and the result will be mutation or deletion through non-homologous end joining or homologous recombination

Question 24

What is the target gene in CRISPR-Cas9?

Answer 24

The target gene can be any sequence that is around 20 base pairs in length. The sequence must be unique compared to the rest of the genome and must be present immediately upstream of a Protospacer Adjacent Motif (PAM). It will make the small guide RNA

Question 25

Protospacer Adjacent Motif (PAM)

Answer 25

Consists of a 3 nucleotide sequence: 5’-NGG-3’, where N= any nucleotide. You will probably have this somewhere in a target gene, as it is necessary for the proper binding of Cas9

Question 26

CRISPR-Cas9 plasmid components

Answer 26

The plasmid should contain the target gene sequence, which makes the small guide RNA. It should also contain the gene for the Cas9 nuclease and antibiotic resistance genes. sgRNA base pairs with the sequence on the target gene, localizing Cas9 and allowing it to make a double stranded cut

Question 27

CRISPR-Cas9 process

Answer 27

1. Cells are transfected with genes for Cas9 and guide RNA
2. The genes are expressed, and Cas9 nuclease binds to the guide RNA
3. Guide RNA base pairs with a sequence in the target gene, bringing Cas9 over with it
4. Cas9 makes a double stranded cut
5. Can be followed by non-homologous end joining, where some nucleotides will be lost and we will lose the expression of the gene (knockout). You can also introduce an antibiotic resistance gene to take this a step further

Question 28

Cas9 nickase

Answer 28

Makes CRISPR-Cas9 even more specific. Mutated so only 1 nuclease domain (RuvC or HNH) is active

Question 29

Paired Cas9 nickases

Answer 29

Will make single stranded cuts in close proximity to one another. This greatly increases their specificity. It is unlikely that 2 off-target nicks will be generated within close enough proximity to cause a DSB. Single stranded nicks, they occur off target, are rapidly repaired with no detrimental effects

Question 30

Off-targets

Answer 30

Sites throughout the genome with a partial homology to the sgRNA targeting sequence. They can be prevented by specific sgRNA design and modification of Cas9

Question 31

Knockout mice

Answer 31

Traditionally, we have relied on homologous recombination for this. DNA is introduced into a mouse cell, and usually inserts into chromosomes at random. 1 in 1000 times, it replaces 1 of the 2 copies of the target gene. Frustrating and tedious process. Embryonic stem cells are grown to introduce an altered version of the gene, and you hope that homologous recombination occurs to knockout the gene

Question 32

CRISPR-Cas9 antibiotic resistance process (3)

Answer 32

1. To take this a step further, you could introduce an antibiotic resistance gene. The cells are transfected with a plasmid containing an antibiotic resistance gene, flanked by the same sequences that flank the target gene
2. Target gene is flanked by the same homologous sequences
3. Homologous recombination occurs, replacing the antibiotic resistance gene with the target gene. Regardless, the target gene is knocked out and the protein is not expressed

Question 33

When are conditional or tissue-specific knockouts used?

Answer 33

When knocking a gene out in an entire organism would be lethal. They use the Cre/lox system

Question 34

Cre/lox system

Answer 34

Uses a floxed gene and Cre recombinase. This system is conditional/tissue specific. Cre recombinase is under the control of an inducible promoter. The promoter may be active under certain conditions (administration of a chemical inducer) or may be active in certain tissues (responsive only to transcription factors present in a specific tissue and not others). A Cre mouse plus floxed mouse is a Cre/lox mouse

Question 35

Cre recombinase

Answer 35

Specifically recognizes the loxp sequences and excises the target gene

Question 36

Floxed gene

Answer 36

A gene of interest (the one you want to knockout) that has flanking loxp sequences. It is generated via recombination

Question 37

Creating a Cre LoxP mouse

Answer 37

Cre mice are made commercially, but you have to create the LoxP (floxed) mouse. You introduce the target gene with the LoxP sequences. The 2 mice are put together in the hope that they reproduce and make a Cre LoxP Mouse

Question 38

Cre-loxP (3)

Answer 38

1. Floxed target gene is flanked by loxP sequences
2. Cre recombinase removes target gene
3. Target gene knocked out, protein not expressed

Question 39

RNA interference (RNAi)

Answer 39

Triggered by the presence of dsRNA- dicer nuclease cleaves dsRNA into small interfering RNA (siRNA), which is around 23 base pairs in length. Argonaute makes siRNA single stranded, and the RISC complex is directed to complementary RNA by single stranded siRNA. The target RNA is degraded by the RISC complex, creating a knockdown. The gene is no longer expressed because its mRNA is degraded

Question 40

siRNA vs shRNA

Answer 40

siRNA is synthesized in a lab and must be transfected at a high concentration. Short haired RNA (shRNA) is delivered on a vector plasmid and can be stably expressed for up to 3 years. Both are used to create a knockdown

Question 41

siRNA

Answer 41

An original siRNA targets RNA for degradation, which results in more siRNA. It is self-perpetuating, as there is continued elimination of target RNA. siRNA may also be passed to progeny cells, allowing for heritable changes in gene expression

Question 42

RNAi in animal models

Answer 42

Recombineering used to make transgenic mice that express shRNA under control of inducible promoter. Similar to Cre system, but mRNA is targeted

Question 43

What is the purpose of site-directed mutagenesis?

Answer 43

More specific than knockouts and helps to determine which parts of the gene are important for function, or to determine the gene/protein domains needed for certain functions. The gene is mutated at a single site in order to study it

Question 44

Creation of site-directed mutagenesis (5)

Answer 44

1. Short, synthetic DNA molecule containing the desired mutation serves as a primer for DNA synthesis
2. Creates a DNA mismatch. DNA repair machinery will come to take care of the mismatch. In some cases, the sequence will be brought back to the original. However, sometimes it will cause a mutation
3. As the cell divides, one progeny will have the correct copy, and one progeny will have the mutant
4. With the right oligonucleotide, multiple codons may be mutated
5. Mutated cells are created

Question 45

Methods of measuring gene expression (5)

Answer 45

1. FISH- degrade DNA and crosslink RNA
2. Reporter genes
3. Quantitative RT PCR (qPCR)
4. Microarrays
5. RNA seq

Question 46

Reporter gene assay

Answer 46

A way to study gene expression, but is really looking at the ability of a promoter to stimulate gene expression. Clones the promoter of interest onto a reporter plasmid, so the promoter is directly upstream of a reporter gene. The reporter gene is easily visualized, like with fluorescence. The stronger the promoter is, the stronger the fluorescence

Question 47

Quantitative PCR (qPCR)

Answer 47

Gives an idea of specific gene expression- quantifies gene expression via measurement of mRNA levels. The first step is RT-PCR, then conventional PCR, where fluorescent dyes are added. The dyes only fluoresce when bound to DNA, which came from the RNA. Fluorescence measurement can deduce the starting concentration of mRNA and mRNA expression levels. qPCR has made northern blotting more or less irrelevant

Question 48

Purpose of DNA microarrays

Answer 48

The microarrays monitor the RNA products of thousands of genes at once. This is helpful in identifying and studying gene expression patterns that underlie cell physiology by looking at groups of genes together- which genes are switched on or off as cells grow, divide, differentiate, or respond to different stimuli.

Question 49

Creation of DNA microarrays

Answer 49

Tiny glass microscope slides are studded with thousands of DNA fragments that we will use to look at mRNA expression. Some are prepared from large DNA fragments generated by PCR and spotted onto slides by a robot. Others contain short oligonucleotides synthesized onto the surface of the glass, using techniques similar to those used to etch circuits onto computer chips

Question 50

Steps in a DNA microarray (5)

Answer 50

1. RT-PCR is first step to make DNA from RNA
2. cDNA labeled w/ fluorescent probe
3. Microarray incubated w/ cDNA – hybridization
4. Array washed to remove unbound cDNA
5. Bound cDNA measured by automated scanning-laser microscope. They are viewed based on color. For example, DNA with increased expression of mRNA shows up as red, decreased expression is green, and no change is yellow

Question 51

RNA sequencing

Answer 51

Arose due to issues with microarrays- cross-hybridization artifacts, poor quantification of lowly & highly expressed genes, needing to know target sequence beforehand. It basically uses next-generation sequencing approaches- cDNA is made first, then subjected to NGS. NGS is performed directly on RNA

Question 52

Direct RNA sequencing

Answer 52

Directly sequencing the RNA without making cDNA. Polyadenylated RNA is captured by poly-T nucleotides, dideoxy-based NGS performed. The RNA can be run through a column or flow cell to enrich it

Question 53

Benefits of RNA sequencing

Answer 53

Facilitates the ability to look at alternatively-spliced transcripts, post-transcriptional modifications, mutations, & changes in gene expression. Instead of limiting analysis to a set of transcripts as in qPCR or microarray, the whole transcriptome is sequenced & measured. Interesting changes/observations should ALWAYS be confirmed w/ qPCR