Sizes in Biology

Dear colleagues,

An excellent tool is available to get a feeling about molecular sizes in Biology.

The tool is available here and was developped by "Learn Genetics" program from the University of Utah.

You can have a short movie on it below, but please check the original one from here!!!








You can also have a deep immersion in the issue here:

Designing a gene or "Gene design"

On this tutorial you can see an easy way to design oligos for gene synthesis, or in other words Gene design.

The description is based on the Instructional Videos from the iGEM website!

Gene design in less than 10 minutes!

Good luck!!!




Some usefull tools:

1. Gene design web page
2. Tools at DNA20
3. Tools at DNA Works
4. A Revese-Complement Tool
5. Gene designer a comprehensive tool to artificial gene design from DNA20 described here.


If you would like to read about the application of design principles in Drug Discovery you should read this book:
 

QPCR oligo design

Designing QPCR oligos might seem complicated but there are some rules and softwares that can make it easy. In our lab, majority of data points we generate are probably measured by QPCR, so I think  it is worth to review an algorithm for desiging QPCR assays.

So, today I will describe the way I design QPCR oligos. You can have a basic intro in PCR here.
More than a year ago I switched to the UPL system, the library of probes designed by Exiqon and now marketed by Roche. The concept is quite clear, the LNA modified oligonucleotides bind much stronger to the DNA template than the average oligonucleotides. By this we can decrease the lengths of them by keeping the Tm unchanged. The UPL library consists of 165 individual oligonucleotides that are in general nine basepair long and together cover the entire genome in respect of the coverage needed to design a QPCR oligo set for any gene. You can read more about the LNA nucleotides here and here. A good website where you can calculate the Tm of the LNA oligos here: http://lna-tm.com/. The UPL system is described here.

The system allows the design of an oligo set for any DNA sequence in the UPL Design Centre. You can access the Design Centre directly here.

[caption id="attachment_581" align="aligncenter" width="468" caption="UPL Assay Design Center"][/caption]

Before we design an assay let us first look for the transcripts of a specific gene. It is very important to use annotated data, since in the annotated genomic data we have informations about possible SNP variations. This might be important, since the oligos and especially the probe should bind to an SNP free free region, because an SNP might disturb the binding of the probe to the template.

To make this data available we should use not the sequence but the transcript ID from the Ensembl.

[caption id="attachment_586" align="aligncenter" width="468" caption="Ensembl"][/caption]

At Ensembl select your species of interest, e.g. mouse and write the name of your gene of interest in the search box:



If you write into the box a gene of interest (e.g. COUP-TF2) we will see the results as it is shown here:



Here I have to click on the link of the gene and I will find the following screen: The most important info we are looking for is in the table on the top of the page:



The two transcripts of the gene are:
ENSMUST00000089565
ENSMUST00000032768.

We will use these ID-s in the UPL Assay Design Centre. First select as organism the "Mouse", and write into the box the two Ensembl transcript ID-s selected by commas. If you follow the steps the results will be like this:

Below this data you can see two links as it follows:To design an assay that would measure all transcripts select: "common assays".

The results will be given in a downloadable pdf report. Save this file and name is by the name of the gene you used as input.

The results in the pdf file look like this:



You can see that the amplicon is 95 bp long, there is no SNP in the binding regions of the primers and probes and the probe is closer to one of the primers. There is an SNP in the gene that was avoided by the program. You can have an SNP even in the amplicon, unless is not in the binding site of the primers or the probe.

Before I order the oligos, I usually test them with e-PCR on the UCSC Genome Browser.

[caption id="attachment_608" align="aligncenter" width="468" caption="UCSC Genome Browser"][/caption]

Select the PCR view and paste the oligos into the given locations. Select the genome, the assembly and the target as "UCSC Genes"(If you used a genomic sequence for design use the target: "genome assembly").

If you have a hit, click on the link provided and the results will be represented in the genome as seen here:



The oligos are intron spanning and in the right location. Order the oligos in an HPLC pufied form in the lowest available scale for the first try. Be aware that according to the experience of several groups, and my own experience too,  only 2/3 of the UPL assays work without further optimization. This means, if you want to be sure from the first you better try two, three different assays for the same gene. The UPL Design Centre will generate several primer-probe sets and you can retrive these results too. Since the UPL library is given and one or two of the three ordered assays will work, it this worth trying three from the beginning!

In general the rules for a good QPCR assay:

1. The amplicon should be as short as possible (60-70 bp is ideal, but should be shorter than 100bp).

2. The Tm of the oligos should be around 60C, while the Tm of the probe 10C higher.

3. The distance between the the oligo and the probe should be as small as possible for a better exonuclease activity of the Taq polymerase.

4. The GC content of the two oligos should be as close as possible.

5. The number of GC bases in the last five nucleotides on the 3' ends of the two primers should be identical (if possible).

6. Select for oligo sets with week internal bonds (less than four H bonds in the same conformation).

7. Avoid primer dimers that could produce artefacts due to the 3' elongation of one of the primers.  The same for internal conformations.  See below:

8. Verify the oligos with e-PCR on the UCSC Genome Browser. The test should give one single hit!

9. If possible use annotated sequences to avoid the SNP effect.

10. If you are looking for genes (cDNA measurement) use exons that are common for all transcripts variants (or use the "batch assay-common assays" in the UPL Assay Design Centre)

Good luck!

Restriction Enzyme Resources

Dear Colleagues,

Sometimes it is good to have links that cover a topic. This is why I have collected here a bunch of links that might be useful for you in your work.

You can have a good description of the methods used in restriction enzyme analysis here: Methodbook.net

If you would like to use restriction enzymes for your work, you can find a list of links of the best known restriction enzyme providers below.

New England Biolabs

Promega

Roche Applied Science: Benchmate

Invitrogen

Fermentas

If you want to start your work with these enzymes, please consult the protocol provided with the enzyme or check it at the website of the manufacturer.

Be sure you know what an isoschizomer is, what star activity is, or how you can make double digestion (details here and here).

Hunting Viruses

If it comes to speak about the future possibilities of molecular biology, it is worth keeping an eye on the medical applications. And if you think about the most peculiar infection agents you should for sure think of viruses also. What is a virus? Of course we all know what a virus might cause to us, like a simple respiratory infection. These are usually caused by viral infections that later are super infected with bacteria. I had a professor who tried to explain us what a virus is. It skips almost any definitions. We can not be sure if we could consider a living thing at all!!! At the end he told us in a laconic way: a virus is a VIRUS! Nothing more.

If we try to find them it is good to know, that they were discovered through the observation that you can transfer an infection from one cell culture to the other even after filtrating the solution through a filter with 0.4 micrometer holes. That means that no bacteria can bypass this filter, but infections can be transferred with this solution. The firs experiments were done in order to monitor these infections, to see that after infection there was a "clean window period", a period when the infections agent disappeared from the cell culture. After this window the virus reappeared and the supernatant solution had infections properties again.

Today we know plenty of details about viruses. There are basically two flavours of them DNA and RNA viruses. So that is an important point! because we have plenty of molecular biology tools that allow us to characterize nucleic acids. One of the most complex tool from this series is the DNA microarray. As one of my students pointed out last week, in the next video from TED, we can have a wonderful presentation about how these tools can be used in a fast and relatively easy way to get a deeper insight in the world of viruses. As a perspective the video shows us some excellent diagnostic applications that will be probably used to develop state of the art diagnostic tools in the next couple of years.

So, let us see how it works!



More info about the viruses and vaccination, here.

Restriction Enzymes

Restriction enzymes are used to cut plasmids. We have tackled the plasmids in the previous lecture. You can have a full description about the restriction enzymes here.

As a most basic introduction I would say that restriction enzymes are enzymes of the bacteria representing a kind of immune function of the bacteria. They are present in pairs in bacteria: a DNA methylase and a restriction enzyme. They both recognize the same sequence. The bacteria is methylating its own DNA in a sequence specific manner. By this its own DNA is protected against any foreign DNA. Since horizontal gene transfer is quite common in bacteria, the bacterial cell can protect its own genetic material with the help of the restriction enzymes. The foreign DNA entering into the cell will present a different DNA methylation pattern. The unmethylated recognition sites will be cut  by the restriction enzymes and by this destroyed.

Different bacterial species have different restriction enzymes with different recognition sites (certainly each has a DNA methyltransferase, too). The nomenclature of the restriction enzyme reflects their origin. In the most trivial case the name Eco RI enzyme is informing us that it has been isolated from Escherichia coli strain R and it has been the first to have been isolated from this strain.

In the molecular biology lab we use them to cut and manipulate plasmids. They are like scissors that can be directed to specific sites in the plasmid to cleave it. With an appropriate collection of site specific cutting enzymes we can step into the very exciting field of genetic engineering.

Let us have a look to some basic usage of restriction enzymes:



You can check a good introductory video here.

In any case when working with enzymes, please use latex gloves, and keep enzymes on ice!

The unit of a restriction enzyme "U" stands for the amount of enzyme needed to cut 1microgram of plasmid with a single cutting site, in one hour, in ideal environment.

The environment of the reaction is provided by buffers. The enzymes are usually provided in a concentration of 10U/ul (10 units per microliter). The enzymes are supplied in glicerol solution and always stored at -20 C. The buffer may as well come in a 10 fold concentrated solution (10X) and it should also be kept frozen.

A typical restriction enzyme reaction is set up in the following way:

1. Check the map of the plasmid for the distribution of the cutting sites.

2. Measure the concentration of the plasmid solution by spectrophotometer. Your plasmid concentration should be in the range of 1 microgram per microliter.

3. Calculate the volume of the plasmid needed to have the required amount of product at the end. The volume of the reaction should be kept as low as possible, and should not exceed 100 ul/ reaction tube. Use sterile, DNAse free microcentrifuge (so called) "Eppendorf" tubes.

4. Plan the reaction. You should have approx 1 to 10 U of enzyme per microgram of plasmid. In the final volume of the reaction the total volume of the enzyme should be less the 1/10, because higher glicerol concentration might alter the specificity of the reaction. The buffer will be 1/10 of the final volume. Keep the final volume low (less then 100 microliters). If needed, adjust the reaction volume to the planned final volume with nuclease free water. Check the optimal temperature for the reaction. It is usually 37C, but it might differ. Check for possible star activity of the enzyme in its data sheet.

Example:

Mix the following components (ul stands for microliter):


16ul Nuclease Free Water+

1ul Plasmid solution (concentration 1ug/ul)+

2ul 10X Buffer+

1ul Restriction Enzyme (10U/ul)


Total:     20ul


5. Once the reaction is planned, start to do it: bring ice, prepare tubes, melt the buffer in your hands.

6. Pipette the required volumes of water, plasmid and buffer into the tube.

7. Add the enzyme to the tube and mix gently. Do not vortex!

8. Put the reaction into the thermostat set to the required temperature.

9. Put the enzyme and the buffer back to -20C and clean up you bench!

10. After the allocated time has  passed, stop the reaction. We are usually keeping the reaction in the thermostat for 4 hours. You can stop the reaction in several ways: by adding EDTA; by heat inactivating the enzyme at 85C for 10 minutes, or simply by freezing the tube and keeping it frozen until you purify it.

You can have a look on the applications in the video below.

Good luck!

Green Fluorescent Protein or GFP

Green lights in the dark



When someone first shows up in our lab, the prime goal I set up for him or her is to make "green cells" - I mean to introduce a Green Fluorescent Protein into a mammalian cell culture. In order to be able to perform this one has to know some basic molecular biology. One has to know what a cell is, what the difference is between a prokaryote and an eukaryote cell; what the central dogma is namelly that the information flows from DNA to RNA and from here to proteins is, or as it has been formulated originally and still correctly, the information flows from nucleic acids towards proteins (albeit I assume we will see exceptions for this rule, too). (You can reach a very good lecture on this topic here.)  One has to know what the difference between DNA and RNA is, in most basic approach the chemical difference is minuscule (there is a deoxyribose in the backbone of the DNA and a ribose in the RNA, there are other differences but this is the most prominent), while the results are spectacular. DNA is a quite stable molecule that can be degraded by DNAses. DNases require divalent metal ions for their activity ( usually Mg, but other divalent ions can be used too), and we can remove these ions from solutions with so called chelating agents. Most commonly we use EDTA for this task.

From practical point of view, one needs to have some backgrounds in order not to be lost in a molecular biology lab as it follows:

One has to be able to use pipettes (as seen in the previous posts), to make buffers, to know about pH, know what molarity is, and have a good basic background in maths (just enough to calculate the compositions of the buffers).

But you can perform the most basic experiment of DNA isolation even in the kitchen! At the end of this experiment you will be able to even SEE the DNA!

You can extract DNA from any cell, but the easiest way is to use some germs, like wheat or bean germs, soya germs and so on... In the following video you can see the procedure. If you do not have isopropyl alcohol (I don't have at home for example) use regular ethanol or a strong spirit with at least 70% alcohol content!



Regarding RNA, the world of RNA is a transient world.  RNA is degraded by enzymes that can be found everywhere. RNAses can not be blocked by removing metal ions with EDTA. This makes the half life of RNA very short. Let us take the analology of the computer: DNA is like the information on the hard disk, one might have a software on the computer without using it- this is the information in the DNA. If one double clicks on its icon, the program starts, this corresponds to the transcription: information is transcribed from DNA to RNA, or the software is running, even if it is not yet in use, it is ready to get an input and process it into the output. The RNA is similarly translated by ribosome into proteins: these are the products that have been coded in the DNA. Or according to the computer analogy you create a document with the word processor software. The document is an entity by itself.  You can print it and have it. If you turn off your computer, the temporary files are destroyed, all unsaved files are deleted. So is with the RNA. RNA is carrying an information for a short period of time, it has a short half life, but can be regenerated from the DNA. These processes are explained in the following video:



Ok, so how do we make green cells? Green flourescent protein is encoded in the genome of the Jelly fish. The protein once identified can be introduced into other organisms if we isolate the DNA sequence that is encoding the GFP protein. So let's have a look to these wonderful organisms!

Beautiful Jelly fish



The discovery of GFP protein and their mode of action changed plenty of studies in biology. The Nobel Prize for Chemistry in 2008 was given for the identification of the GFP protein and its way of action. You can see below two videos about the topic. A detailed, in depth one or below a short overview of the topic. You choose!

Giving green light to biology



Nobel Prize for GFP



After this overview I think it is time to have an experiment. We will see how you can introduce the GFP encoding DNA into a bacteria. For this we use so called plasmids as a vector. We call vector in biology a tool that is able to carry genetic information, like a plasmid, cosmid, or a virus. A plasmid is a small circular DNA that is able to self-replicate into a bacteria and to express a protein. They are responsible for lateral gene transfer in bacteria, e.g. transfering antibiotic resistance gene from one bacteria to a different one.

In the following experiment we will see the introduction of a GFP encoding DNA into a so called Agrobacterium, a bacteria that is infecting plants.

Introducing the GFP into a bacteria



Cool, isn't it?

We can make even more complicated investigations with the help of the GFP. In the following animation it is shown the transfection process in a mammalian cell where the addressed question is if two proteins interact or not? For this they use the so called FRET or fluorescence resonance energy transfer. In order to see if the two proteins are close to each other or not, we have to use two GFP like tagged proteins with their excitation and emission wave lengths close to each other. See how it works:

Investigating protein-protein interactions with fluorescent proteins



GFP has several other applications, like tracing of migrating neurons, as seen in the following video:



Or full GFP organisms like in the following one:



If you would like to know even more about the GFP protein, please visit the best site in this topic I have ever seen, the page of Marc Zimmer, here.

I think we had even too much of GFP now, so in the next posts we will go back to plasmids...

See you!

Why Molecular Biology?

At the very end you might ask why is the life in a molecular biology lab so interesting?

We discussed about water, pipettes and we will go on with several topics, but at the very end there is a wonderful, miraculous world. Each cell in our body and each cell in any living organism works based on the same principles. Information is stored, processed and replicated in cells.

If we could have an insight into these processes we could better understand what is life. Yes, I think this is still a question! What is life? How can you explain the abundance seen on every cubic centimetre of the surface of this planet?

Instead of giving a flat answer, let us look to the best animation I have ever seen about THE INNER LIFE OF THE CELL!

Here it is:

Dear all,

The setup seems to be quite clear.

Every semester we have at least five students coming into our lab to get involved in molecular biology techniques. Untill now, those who started their training with me, all had to go through the same basic steps. I will follow this method and we will write comprehensive protocolls about these steps.

The basic route in starting to work in our molecular biology lab was the following:

I. INTRODUCTORY PART

1. Introduction to the members of our lab.
2. Safety rules and regulations in the lab.
3. How to deal with the garbage, trash and other materials produced during the experiments.
4. How many tipes of water do we use?
5. What kind of reagents to we use?
6. Where and how do we store our reagents and our samples.

II. BASICS IN THE MOLECULAR BIOLOGY LAB

1. How can we protect our sample from degradation? If our sample is:
a. DNA
b. RNA
c Protein

2. DNA purification methods.
3. RNA purification methods.
4. Quallity control (QC) of DNA and RNA

III. BASIC TECHNIQUES TO GET USED TO LABWORK IN THE MOLECULAR BIOLOGY LAB

1. Working with plasimds:

a. Transformation
b. Growing of bacterial culture
c. Plasmid purification
d. Restriction analysis
e. Gel electrophoresis

2. Working in the cell culture lab:

a. Cell culturing basics
b. What is "sterile" in the issue culture environment?
c. Adherent and floating cells.
c. Making a passage.
d. Cell counting.

3. Transfection

a. Our methods used for transfection
b. QC
c. Sample preparation

4. PCR

a. Introduction into PCR
b. PCR and QPCR in practice
c. Analysis of the results

IV. ADVANCED TECHNIQUES IN THE MOLECULAR BIOLOGY LAB

We will elaborate this section later. We plan to include Western Blot, Protein purification, Modility Shift studies, Chromatin studies. etc.

Dear Colleagues!

I decided to make an online collection of the basic (and not so basic) techiques we use in our lab. This is a (hopefully) classical molecular biology lab located in Europe, Hungary, more close in Debrecen. I will present you the lab and environment later.

The idea is to describe these techiques, make a pdf version of the protocolls we are using and in some of the cases to upload videos about these techinques.

Any feed-back is wellcome.

Please stay tuned,

Balint