Lecture 4: basic techniques

Question 1

How can you isolate DNA from cells (4 steps)?

Answer 1

1. Prepare cell extract
2. Remove other cell components (proteins, lipids, RNA) by treatment with detergent and/or organic solvent
3. Precipitate DNA with alcohol (ethanol or isopropanol)
4. Dissolve in buffer solution

Question 2

What is often used for specific binding of DNA?

Answer 2

Porous material

Question 3

What technique is used to seperate DNA fragments and how does this work?

Answer 3

Agarose gel electrophoresis. Here porous gel is used to seperate the DNA. A positive electric field is created over the gel that attracts/pulls the DNA fragments apart. The mobility of the DNA is then dependent on the lenght/size of the DNA fragment.

Question 4

How are DNA fragments made visible during agarose gel electrophoresis?

Answer 4

The gel is stained with DNA-binding dye such as ethidium bromide.

Question 5

Purified DNA can then be split by enzymes cutting at specific sites. How are these enzymes called and how are they able to cut DNA?

Answer 5

Restriction endonucleases. They recognize specific recognition sites of 4-8 bases. These sequences that are recognized on the double stranded DNA strand are palindromes.

Question 6

Purified (and/or restricted) DNA can also be connected to other DNA fragments. What protein is needed?

Answer 6

DNA ligase

Question 7

Purified DNA can also be transcribed into mRNA and translated to yield a protein. What is needed for this?

Answer 7

RNA polymerase and ribosomes (so everything that is needed for transcription and translation). This is usually performed in a suitable host cell (can also be done in vitro).

Question 8

Purified DNA can be amplified to have enough material for your application. What technique is used for this?

Answer 8

Polymerase chain reaction (PCR)

Question 9

Sum up the materials needed for polymerase chain reaction (PCR).

Answer 9

DNA fragments to amplify, primer, nucleotides, buffer, DNA polymerase (stable enough to withstand heat), PCR machine (thermocycle)

Question 10

There are 3 steps in polymerase chain reaction (PCR). Name them.

Answer 10

1. Denaturing (seperation) of the double DNA strands by increasing the temperature.
2. Lowering the temperature so that the primer can bind to the single stranded DNA.
3. DNA polymerase that makes the new strand

Repeat cycle (20-40x)

Question 11

For what do we use reverse transcription PCR and how does it work?

Answer 11

E.g. to investigate mRNA in a given cell. Here, reverse transcriptase is added to synthesize DNA from already existing mRNA. After this normal PCR can be used to amplify the synthesized DNA.

Question 12

For what do we use real-time qPCR?

Answer 12

To amplify and quantify DNA at the same time

Question 13

There are two mechanisms that can be used for real-time qPCR. Name both of them.

Answer 13

1. DNA is quantified by binding of fluorescence dyes that bind to double stranded DNA. More DNA also means higher intensity of fluorescence.
2. During classic PCR a probe is added to the mixture with the denatured strand. The probe contains a fluorescent signal, but also a quencher. The quencher dims the fluorescent signal if they are close to each other. When DNA polymerase makes the new strand it cuts off the probe by cutting it in tiny pieces. This causes the quencher and fluorescent signal to be distant from each other, which increases the fluoresence intensity. See picture for 2.

Question 14

There are ways to amplify DNA without thermal-cycling. This, because thermal-cycling can be quite expensive. An example is the use of Nucleic Acid Sequence-Based Amplification (NASBA) for detection of RNA (viruses). How is this technique performed?

Answer 14

1. Reverse transcriptase makes DNA from RNA.
2. RNAse H degrades RNA bound to the DNA
3. RNA polymerase akes new RNA -> use for step 1

Question 15

What is site directed mutagenesis and why would you use this?

Answer 15

Introduce point mutations into the DNA. This way you can investigate its effect on the protein, e.g. which role the changed base plays in the function of the protein.

Question 16

How can you make a site sirected mutagenesis?

Answer 16

You first denature the double stranded DNA template. Next, you anneal a mutagenic primer containing the desired mutation and let DNA polymerase make a new strand from the primer on. (Usually done with plasmids that are then inserted in the host).

Question 17

What is the purpose of Sanger sequencing and how does it work?

Answer 17

The purpose is to generate every possible length of DNA up to the full length of the target DNA. This is done by starting with performing PCR on the DNA strand of interest. After this all the double stranded DNA are denatured and a oligonucleotide primer is added. The DNA strands with the primers are divided into 4 tubes, these tubes contain normal nucleotides and ddNTP nucleotides. When ddNTP nucleotides are added (at random basepairs) to the strand by DNA polymerase, the replication process stops. This results in multiple different lenghts of DNA fragments.

Question 18

Why do we purify proteins before investigation?

Answer 18

To investigate proteins without contamination (other cell components) or to use or sell proteins.

Question 19

What is column chromatography? Also name two examples of column chromatography.

Answer 19

A method to purify protein where you pass a sample through a column filled with porous material. If and how fast protein will move to the porous material depends on the type of protein. Examples are gel filtration and ion-exchange.

Question 20

What is gel filtration?

Answer 20

Certain gel beads are used as column material with narrow pores. Seperation is then dependent on protein size where the large proteins are the first to be eluted.

Question 21

What is ion-exchange?

Answer 21

Here a column material is used with a charged surface where seperation is now based on protein charge. Proteins without a charge or with the same charge as the column elute first.

Question 22

What is polyacrylamide gel electrophoresis (PAGE) and how is it performed?

Answer 22

The purpose of PAGE is to purify, seperate and determine the size of proteins. PAGE is performed by adding a negatively charged detergent (SDS; sodium dodecyl sulfate) to the protein sample to give them a certain negative charge. When a charge is created over the gel, the proteins move to the positive node. But some proteins move more slowly because of their larger size. Next, you can stain the proteins to visualize them and you can calculate the size of the proteins.

Question 23

What protein is most commenly used to label proteins?

Answer 23

Green fluorescent protein (GFP) (there are also other variants of GFP).

Question 24

What is co-immunoprecipitation (i.e. what purpose does it have and how does it work)?

Answer 24

The purpose is to find protein interaction partners of the protein of interest. First you make a cell or organelle extract, add the antibody against protein of interest. And lastly test which other proteins are precipitated.

Question 25

What purpose can knock-out mouse models have?

Answer 25

The purpose is to gain information on what function a gene has (what function is lost if you knock out a gene?). The genes that are studied can be involved in obesity, cancer or other diseases.

Question 26

How is a knock-out mouse model made?

Answer 26

1. Culture embryonic stem cells 2. Transfect these stem cells with DNA carrying a manipulated/interrupted gene. 3. Carry out homologous recombination 4. Insert in mouse blastocyst

Question 27

Beside transfecting the embryonic stem cells with DNA carrying an interrupted gene during knock-out mouse, what else is important to add in the DNA?

Answer 27

A resistance marker, so you can distinguish the embryonic stem cells that have taken up the proper DNA carrying an interrupted gene from the ones who didn't take up the proper DNA.

Question 28

Inserting DNA carrying an interrupted gene for knock-out mouse models doesn't mean that the first mouse you get, is a knock out mouse. How do you get this knock out mouse that is carrying the gene that has been knocked out?

Answer 28

So when the DNA is insterted in the blastocyst, the blastocyst is put back into a 'foster mother'. A chimeric mouse is then born that is then crossbred with a 'normal' mouse. This way you get a heterozygous mouse for the gene knock out. You can then crossbreed this heterozygous mouse with another heterozygous mouse for the same gene knock out, to get a mouse that is homozygous for the gene knockout.

Question 29

Sometimes the knock-out mouse model doesn't work, for example when the gene you knock out has a lethal result for the mouse. What technique is there to bypass this problem?

Answer 29

RNA interference, here gene expression is silenced instead of knocked out.

Question 30

How does RNA interference work?

Answer 30

Double stranded RNA is cut into small interfering RNAs (siRNAs) by the enzyme dicer. siRNAs bind RNA-induced silencing complex (RISC). RISC separates the double stranded siRNAs, where the guide strand of these RNAs subsequently bind to a complementary target sequence. This ensures that translation of the target sequence of RNA cannot occur, thus the gene is silenced.

Question 31

What can you use in RNA interference to increase the time a gene is silenced?

Answer 31

The use of plasmids that code for siRNA

Question 32

How can you use RNA interference to silence ATP synthase gene expression?

Answer 32

You can use siRNA that is complementary to the mRNA for ATP synthase subunit c (key part of ATP synthase)

Question 33

Consider the situation where a plasmid is constructed for the production of siRNA against ATP synthase gene expression, where the transcription of siRNAs is tetracyclin-dependen. Next, the plasmid is transformed into bacteria where RNAi production is activated with tetracyclin. Are the bacteria then still viable?

Answer 33

No, so there's a tetracyclin-dependent decrease of bacterial cell density.

Question 34

What are limitations of RNA interference?

Answer 34

That there are off target effects we don't know (is it really only targetting the mRNA of interest?). And also, without the use of a virus as a vehicle, there are only short-lived effects.