Lecture 14 (RR1): Nucleic Acids: from detection to quantification Flashcards
How can molecular biological techniques advance our capacity for analysis?
Techniques can help us do two types of analysis: qualitative analysis and quantitative analysis.
QUALITATIVE ANALYSIS.
- the nature of the molecule(s) in question
- size
- nucleotide composition? (the sequences of specific genes)
- conformation/configuration? How various conformations can change
- 3D structure that macromolecules may occupy in space?
QUANTITATIVE ANALYSIS → important in diagnostics
-determine the levels of gene products.
ie…tumour markers (p53, BRCA1/2)
Molecular Probe
1) Its use?
2) How to use it?
1) A molecular probe allows us to detect specific nucleotide sequences and we can detect these sequences down to a picogram level. It is highly efficient.
2) - start with a mixture of macromolecules
- run the macromolecules through an electrophoretic field. This will seperate the macromolecules by size.
- Transfer molecules to solid-state (creates a permanent seperation)
- Hybridization: the reverse compliment of the probe will pair to the nucleotide target sequence( watson crick base pair) –> black triangles on diagram. The probe will interact nonspecifically with all the nucleic acid macromolecules. You can remove that non-specific interaction by washing the solid state membrane with buffers and increasing the temperatures. But if it is 100% complementary, it will not let go.
- This allows you to reveal the position of the target nucleotide sequence and get an accurate idea of the abundance of that particular sequence.
How do you make an oligonucleotide probe?
Take the oligonucleotide that you prepared and subject it to a PNK reaction. Polynucleotide Kinase (PNK) will phosphorylate nucleotides by transferring the γ phosphate of ATP to the free hydroxyl at the 5’ end of the synthetic oligonucleotide. It is 5’ end labelled. The gama phosphate has either been modified to be either fluorescent or radioactive.
How can you use PCR to make labelled DNA probes?
- PCR can be used to make DNA probes because PCR will incorporate nuclotides into a growing polymer.
- Make an upstream and downstream primer that you would use for any PCR reaction.
- Modify the nucleotide mix: diminish the concentration of one of the nucleotides (dNTP) and add a labelled variant of the same nucelotide you diminished (fluorescently or radioactivaly labelled)
- Has to be labelled on the alpha phosphate!!! the alpha phosphate is going to be retained and incorporated into the polymer.
- Polymerase will grap the labelled nucleotide (every once in a while) and put it into the polymer as it is synthesized.
- Last step, purify the amplicon and then render it single stranded so that it is ticky and can interact with target sequence - must denature before using.
Southern analysis
- Both DNA and RNA can be separated according to size using an agarose gel.
- DNA is cut with a restriction enzyme. DNA is cut –> small enough to go into the agarose sieve. RNA, will go into that agarose gel, but still denature RNA –> avoid folding up in weird structures. Denature to assure they will seperate based on size.
- Run through an agarose gel. It will seperate the nucleic acid molecules acording to size
- Put them onto a solid state support, nucleic acids bind strongly to the membrane (can be permanently bound by UV crosslinking) → to do this:
1) after the electrophoresis, denature the DNA in the gel by putting it in an alkaline bath.
2) Use some alkaline based buffer to draw up the gel and bring the DNA molecules onto this nitrocellulose (diffusion - pretty much a towel on top that absorbs all the water and binds to the fragments - RNA or DNA). This permanently records the levels (abundance) and the position (size) of the molecules following separation on the gel.
3) Allow this to interact with your molecular probe.
4) To hybridize with labelled DNA or RNA probes to any sequence that may be of interest, use a wash. Washes remove non-specific signal and only complementary sequences will be detectable on the blot following autoradiography
How do you detect polymorphisms with probes?
- Remember: probes do not have to bind 100% of their gene targets.
Gene Z harbors the nucleotide sequence that we are interested in. - We have a probe that recognized the region.
- Within gene Z there are 3 restriction enzymes.
- If we were to cut the DNA sample with EcoR1, it would give rise to 2 fragments that would both be recognized by the probe.
- If we ran this on a gel we would have one larger fragment corresponding to fragment 1 and a shorter fragment corresponding to fragment 2 using our molecular probe.
- The probe does not need to bind the entire gene, it can still bind this little but and give you the information needed.
BUT if we have a mutation in Gene Z
* Perhaps the EcoR1 site is modified so that the enzyme no longer recognizes its digestion site.
* So, when you try to cut with EcoR1, it does not recognize the DNA sequence and suddenly you don’t have two fragments that the probe would recognize.
* You have one large fragment that is a combination of fragment 1 and 2.
* The probe will only recognize one large fragment. Therefore, there will be a difference of either one single nucleotide or a few nucleotides that disrupt that EcoR1 site. This will mess up the ability of that restriction enzyme to recognize its site and will give rise to a completely different signature that will give rise to your probe.
What is a technique that can be used for DNA detection?
When you are doing pedigree analysis, you are looking at complex DNA samples from individuals and then you want to compare them to see how related they are or whether certain variants are present or not in each one of these individuals.
- In this situation the probe has been designed to recognize a specific gene sequence that might be associated with the disease gene or some other polymorphism.
Southern analysis can be used to identify polymorphisms:
* The probe recognizes 3 different variants here. The variants are referred to as polymorphisms and they are very often associated with variations of a given gene. They might be alleles in a population. The variants correspond to the DNA sequence of one of the chromosomes in each one of those individuals.
1) The DNA from an individual is subjected to a restriction enzyme digest
2) DNA is ran on electrophoretic field side by side and then probed with a given probe that recognizes a nucleotide sequence of some gene variant (different alleles) that we are interested in
In the parents, one chromosome has a polymorphism. These somehow give rise to this higher molecular entity that is recognized by the probe. So the probe is telling us that there are differences. We don’t know what those differences are but they’re different and we can see how they segregate based on the southern blot.
If you use a probe to detect polymorphisms and you notice that there are differences in what the probe is detecting, what do you do?
- You can amplify the region that corresponds to that gene. Within the amplicon you know that there are specific enzyme sites.
- So by cutting with a given enzyme, you’ll get a characteristic signature based on the electrophoresis that you will do afterwards.
- In this example, we get a small fiber and a long fiber because EcoRI is the appropriate site.
- In the case where EcoR1 is mutated, it can no longer cut that amplicon (amplicon = the region you amplify with PCR). SO, when you run it on a gel, the stained gel shows that there is no cut there (means that EcoR1 did not recognize that the sequence has changed in the region).
- Sometimes this can be diagnostic (sometimes it is a single nucleotide change that predisposes you to this risk factor). Single nucleotide changes are really important and we can detect them very simply by carrying out these signature diagnostic restriction enzyme digests of amplicons of nucleic acid DNA that we know.
What is a technique that can be used for RNA detection?
Nothern Analysis: to find how where the mRNA is expressed and how much?
* Make sure it is denatured, run RNA through agarose gel to reduce complexity, transfer RNA onto nitrocellulose filter/membrane (capillary action).
* Hybridize the membrane with your nucleic acid probe that recognizes that RNA sequence. And then wash to get rid of all non specific probes.
* You end up with these big black spots (radioactive). All of these lanes correspond to total RNA that was collected from each of these tissues.
* You can see some things are expressed more in one tissue than another.
* In order to do a northern blot, you have to make the primers that are specific to that target, so it gives you a high resolution and accurate estimation of the RNA that is there.
- Northern analysis is very accurate and is focused on a single gene product based on your probe.
- It can tell us about the various isoforms of mRNA that is due from, for example, alternative splicing.
- Can give you information on temporal control and regulation. Ex: Sometimes genes get turned on at different times of development - the transcript is on quite strong in the kidney in the fetus but off in the adult tissue.
- It gives you quantitative, qualitative and temporal information.
How to make a cDNA library?
1) mRNA is converted to complementary DNA (cDNA) by priming the poly A tail with a single-stranded poly T oligonucleotide.
2) RT uses this primer to initiate single-strand DNA synthesis that is fully complementary to the mRNA template. At the end of the reverse transcriptase reaction, you end up with a hybrid (green and red strand, the red beam is the mRNA and the green is the cDNA that was synthesized by the RT).
3) RNA is then removed using an alkaline buffer or RNAses that will degrade the RNA. To go from a single stranded cDNA to a double stranded DNA, a poly dG adapter is annealed to the 3’ end.
4) We prime the second round DNA synthesis with a polydC primer. A poly dC primer is used to initiate synthesis of the second DNA strand.
5) E.coli DNA polymerase I progresses through any remaining hybrid regions and extends the second strand. We also include the varying abundances of the various mRNAs.
You end up with a double stranded DNA molecule, a cDNA that corresponds to the sequences of all the mRNAs that were present in that initial sample. You can use cDNA’s that have these identical ends (either all G or C on the 3’end or all T or A on the 5”end) and clone them into vectors. Eventually, you can clone into or insert into bacteriophage or bacteria through infections or transformations and then you cankeep those bacteria in the freezer for almost forever.
A single bacterium will only have one single cDNA associated to it.
Quantitative RT-PCR (RT-qPCR)
In order to do an RT-PCR, you have to make the primers that are specific to that target, so it gives you a high resolution and accurate estimation of the RNA that is there.
RT-PCR relies on doing a reverse transcriptase reaction before you carry out a number of PCR cycles that will amplify the DNA that is made from the cDNA template that you started with.
RT reaction will convert all mRNA to cDNA. This reaction can then be subjected to a PCR reaction that includes a fluorescent dye that emits a signal when incorporated into the growing DNA polymer (the fluorescent dye will only fluoresce when it can interpolate into the DNA that’s being made). So, the more DNA you produce the more dye will intercalate in (more DNA = more fluorescence).
The more DNA produced; the more fluorescence signals are detectable. This can be measured in real time.
* You are restricted to one single gene target, you cannot understand what is going on in the entire genome.
Explain the phases of PCR reactions (graph)
- All PCR reactions go through an exponential phase, a linear phase, and finally they reach a plateau.
- The plateau is reached much faster (in fewer cycles) in samples with greater amounts of starting material (cDNA). Time to reach the plateau is directly proportional to the mRNA abundance in the original sample (dependent on)
- The amount of starting material can be calculated by monitoring the number of cycles it takes to reach the plateau phase.
RNA-seq
RNA-seq is a method for studying global gene expression.
1) Extract the DNA from the sample (you can look at mRNAs, protein coding genes or non coding genes).
2) Let’s say we are looking at protein coding genes. We could do a poly A selection by taking the total RNA that you’ve extracted from tissues and then subjecting them to an affinity chromatography step. You can purify the poly A mRNA because if you run it through a poly T column the poly A will stick to the column.
3) Once you’ve got your mRNA, you can do your reverse transcriptase reaction and make cDNAs (cDNA libraries based on all the mRNAS that were present in your sample).
4) Once you have the double stranded cDNA, you can adapt them with next generation sequencing adapters. These are chunks of DNA that help for the sequencing processes.
5) Subject the cDNAs to next generation sequencing. The number of sequence reads that you get for any given gene product that was present in the cDNA library will correlate to the abundance of the mRNA that you initially had in the sample. Gives you a reasonable estimate of how much mRNA was actually accumulating in the tissue at the time of RNA extraction and you can get this info for every single transcription unit (what genes were on what genes were off at any given time in any given tissue).
- RNA-seq makes cDNA libraries that correspond to all the mRNAs that are present in a sample and then combines that with next generation sequencing technologies.
- Allows us to evaluate the levels of each one of those cDNAs based on the number of times that they get sequenced and realign those sequences to the genome in order to understand how many times a given gene was expressed.
Similarities of RNA analysis by Northern blots and RT-qPCR
- both accurate but really focused on specific gene products transcripts
- Only do them more or less one at a time
- High resolution, restricted breath
RNA-seq vs RT-qPCR vs next generation sequencing
RNA-seq:
* Medium resolution but extraordinary breath
* You get a full snapshot of all of the genes and how they are being expressed in a particular sample
* allows you to see the isoform specific stuff
* RNA seq is quite expensive
RT-qPCR:
* a quantitative method for assessing the abundance of an RNA in a sample.
* Pretty accurate but it does not give you a global idea of all the RNAs (you would have to do 20000 different RT-qPCR instead you could do it all in one experiment with RNA-seq)
* RT-qPCR is not expensive
Next generation sequencing
* Also called deep sequencing
* Around the early 2000s, there was a revolution where you could basically just add on these primers and you could sequence everything at once.
* You sequence everything and let computers put it together. Made things way faster → do not need much information.
* Can sequence from anything.
Use these techniques to understand how specific transcripts are accumulating or the abundances or even the kinds of transcripts that are there.
