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Lecture 4

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Biology (Sci)
BIOL 301
Nam Sung Moon

White colony doesn't mean you have insert+vector! It just means you have something disrupting LacZ sequence. Thus, you need to actually confirm that those colonies actually has vector and insert. There are few ways to do this: 1) Colony PCR: take your pipet tip and touch white colony and throw it into PCR reaction mix. You design 1 primer that will recog insert sequence and the other that recog vector (see hand-drawn pic on the side). This will not only tell you if you have insert, it will also tell you the orientation of your insert (if insert got put into the vector in the other direction, the PCR will occur in the other direction --> won't get PCR product) Once have a clone, we have to ensure that the Biology 301- Molecular Biology Lecture 4: clone has the right insert Gene expression & transcript analysis ➢  Selectable marker & insertion marker only tell you that you have a plasmid with an inserted piece of DNA… ➢  Cloning confirmation ▪  PCR, restriction digest, and sequencing ➢  Three ways to check: ➢  Study of gene function 1. Colony PCR ▪  Determination of protein function through bioinformatics •  PCR directly on bacterial colonies from plate of transformants ▪  Gene expression analysis – transcript level •  Gene specific primers tell if right gene •  Datamining •  Gross tissue-based techniques •  One vector-specific + one gene specific primer tells you if in •  RNA detection directly in tissues right orientation •  Caveat: can get false positives b/c PCR is very sensitive. If you happen to touch colony next 1 to the colony you picked, you can actually have two diff colonies. 2 Context paper: -Nb. we are doing cloning for molecular complementation test. But really, cloning is done for any manipulation of DNA -in context paper, they used Kpn and Nco1 to digest BAC clone that contains sl1 gene (the WT gene) and cloned it into E. coli first (it is 2) Restriction enzyme digests - the one we will use easy to do!). Once they got good clones, they transformed it into -since you know sequence of insert + vector, you can predict Agrobacteria (b/c it is the agrobacteria that will transfer this sl1 gene plasmid sizes if you treat with restriction enzyme. There are 3 types sequence into the plant). of digests ppl use: 1) linarization = chose enzyme that will only cut -in context paper, they made 10 independent rescue lines and they once in plasmid so that the size will be size of insert + vector; 2) cut found that 8 of them rescued the phenotype. They made 10 rescue out the insert = use same enzymes you use to prepare insert + lines b/c there is random integration into plant genome so it could vector --> should get 2 bands (1 = insert and 1 = vector); 3) potentially affect endogenous gene and affect phenotype. Another asymmetric digest = chose enzyme that will cut asymmetrically in possiblity is that gene might get inserted into a part of genome that is vector sequence and in insert sequence (see handdrawn pic below) normally silenced - in this case, the rescue construct will not be --> this will also tell you orientation of insert expressed. Once have a clone, how ensure have right insert? 2. Restriction enzyme digests •  If know sequence of insert + vector, know where restriction ➢ Kpn/Nco1 digested from a BAC clone sites are in both – can predict sizes of bands from digests •  Useful digests (good to do more than one) ➢ Cloned into pCAMBIA (plant specific) –  Linearize – single cut – 1 band expected size of insert + vector ➢ Transformed into Agrobacterium –  Cut out the insert – 2 bands, one size of insert, one size of vector –  Asymmetric – results vary on what enzyme choose – can use for confirmation of sizes and orientation •  Need to do plasmid preparation from bacteria first 3) Sequencing: ➢ Transformed plant using Agro carrying the rescue -good idea for any clone (just to make sure there is no mistake) construct. (Methods of transformation varies among species). -sequencing is very important if you cloned PCR fragment (b/c PCR can have some mistakes due to polymerase) ➢ Eight of ten lines rescued phenotype 3. Sequencing (Control for the location of the integration) •  Good idea for any clone •  Very important if cloned PCR fragment 3 4 the fact that 8/10 lines rescued phenotype shows that this is the gene responsible this is if insert was inserted in the opposite orientation Summary: In doing what is mentioned in previous slide, you will end up with novel genes that you know nothing about. You can then ask several questions -we first did mutagenesis to find mutant that has defect in the process we are interested in about the gene: -with the mutant, we use molecular markers to figure out where gene is. -what does it encode? Is it a TF, a kinase, a part of microtubule..etc? -what happens when it is mutated? If we started with the mutant (this is what -after we have candidate genes, we can use molecular we did), we know the answer to this already. But sometimes, we start with a complementation test to confirm the gene you think is mutated and is responsible for phenotype gene -- in this case, we can inactivate this gene and see what happens = reverse genetics -when/where gene is expressed in organism? We can ask this in terms of transcript (where it is expressed) or where the proteins are expressed. (cont Have identified & cloned the gene, now what? below) gene What is the role of the gene? Normal mapping Molecular complementation ➢ What does it encode? ➢ What happens when it is mutated? ▪  If started with mutant (Forward Genetics), know this ▪  If started with gene (Reverse Genetics), need to create ➢ Where & when in organism is it expressed? mutagenesis ▪  Transcript level shorty cloning the gene for rest of class, we will look at this ▪  Protein level ➢ Where is it found within the cell? ➢ With what does it interact? ➢ How is it regulated/modified? 5 -where is protein found in cell? Does it go to ER or does it go to 6 golgi...etc? we can have some idea of what the protein does based on where it goes -what does protein interact with? -how is it regulated? Is it phosphorylated...etc? When you first get gene and you don't know what it's doing, first thing you can do is you can take advantage of bioinformatics. Bioinformatics compare nucleotide sequence and protein sequence you got with database of Once you have idea of what gene's function is, you can ask where and when it is expressed in the organism. we will focus on known sequences. What you're hoping is to find homology with your gene with a similar gene in another organism that transcript level (RNA) expresion has already been characterized (b/c genes that look similar -first thing you can do is datamining - there are databases that you can look at to get idea of where gene is expressed must have similar functions. If you know about its homologous gene, you can draw some conclusions about -then, experimentally, you can do 3 things (listed as #2-4 in slide) your gene of interest) today, we will talk about #1-3 in slide What does the gene encode? What is the role of the gene? ➢  Bioinformatics – compare nucleotide sequence & predicted protein sequence with database of known sequences ➢  Where & when in organism is it expressed? ▪  Look for “homology” – similar genes/proteins in other organisms ▪  Transcript level and RNA expression pattern that have already been characterized (Blast search, etc) maize each box 1.  Datamining – quick way to get an idea where to black cottonwood Arabidopsis are protein look (need to back up with specific techniques) tomato domains grape 2.  Gross tissue-based techniques 3.  RNA detection directly in tissues 4.  Promoter-reporter gene fusions slide shows results form sl1 context paper. Sl1 is from rice. 7 8 They sl1 from rice and did search and looked for genes that look like sl1 --> they found some similar gene in other plants (arabadopsis has a homologous gene called JAG). So whatever sl1 does in rice could be similar to what JAG does in arabadopsis. Each box in slide is a protein domain. These searches look for protein domains Proteins functions as a domain. We seecertain protein sequences & know what they do (eg zinc finger domain binds to DNA). JAG is a TF --> can predict that sl1 is likely to be a TF (but still need to do experiments to prove this) Datamining -we have these databases as byproduct of large scale functional in the old days, ppl generated EST libraries/databases -how it works: if you want to know what genes are expressed in genomics projects. What they are interested in is not really function of a arabdobsis flower, what you do is you take RNA from flower and convert it single gene; rather, they are interested in genes that are expressed in certain context (eg what are genes expressed in prostate cancer). to cDNA and clone it (ie use cDNA pools as insert to ligate it into vector). Then, sequence it to see what cDNA is present in flower. Problem is, -the problem with these techniques is that they are not designed for when you're sequencing it, you don't know what you cloned - you don't specific genes. You can use it as a guide to generate a hypothesis; but it is never a method to demonstrate something. Thus, whenever you get know sequence info of insert. What they did was use primers that bind to vector sequence (you know vector sequence) and sequenced into the info from datamining, you need to confirm it with more specific tools insert. They ended up getting end sequence of cloned cDNA (EST) (ie got Datamining for gene expression information the ends of the gene sequenced). (cont below) Datamining sources for gene expression ➢  EST libraries/databases - “Expressed sequence tags” ▪  cDNA made from various tissues/conditions ▪  End-sequenced ▪  Not quantitative, but can give you information on tissue where the GOI is expressed ➢  Byproduct of large-scale, publicly-funded functional genomics projects ➢  Databases available on the web ➢  Techniques used are not designed to ask specific questions * Recent advance in technology allows direct ➢  Use it as guide to generate idea, need to confirm with more sequencing of cDNA specific tools 9 To know what is really expressed, you don't have to know the full 10 what are the techniques used to generate these databases? sequence; lots of organisms have their genome sequenced so just having bits of sequence can tell you which gene was cloned. These data -see next slide have been accumulated and there are databases for this. This is how they figured out what gene was expressed in the old days This method is not quantitative. But if you find a particular gene in a Ppl don't use technique mentioned in previous slide particular library, then it must be expressed there (ie expressed in the anymore. But the libraries are still there and ppl use those. area of interest of that particular library) Something ppl still use is microarrays -it is a little chip with probes that are present in specific Usually, people use microarrays to have an idea of the relative expression level locations on the chip. Each location recognizes a particular of the whole genome (this is used to compare expression level) gene. In a single chip, you can have thousands of probes, -for example, you want to compare expression of genes in flower and in leaves. allowing you to analyze the whole genome of the species. You will RNA from flower and leave and make cDNA and flourescently label -you have cDNA that are fluorescently labelled which you them. Then, you hybridize to the microchip that can detect the whole genome then hybridize to this microchip --> particular gene will bind and compare the chip that hybridized with cDNA from flower and the chip that to particular locations that correspond to the probe that are hybridized with cDNA from leaves. You compare the intensity. If a particular spotted gene is highly expressed in flower and not leave, that particular spot will be brighter in the chip that used cDNA from flower Datamining sources – Microarrays (DNA chips) Datamining sources – Microarrays (DNA chips) ➢  Hybridize fluorescently labeled cDNA from tissue/condition/mutant of interest to see level of gene expression ▪  Then can compareresults from chips hybridized with cDNA from different tissues ▪  Need to be confirmed by more specific tools ➢  Many microarray results are publically available Affymetrix oligonucleotide chip (e.g. Arabidopsis ATH1 24K whole genome) ▪  e.g. AtGenExpress - >100 tissues & ➢  Slides dotted with arrays of cDNAs or oligonucleotides– 1000’s of them developmental stages and each spot can recognize a gene!!! ▪ labeled cDNA from tissues of interestat once by hybridizing fluorescently 11 12 ▪  Semi-quatitative there are databases that contain microarray data If there are more of a particular gene expressed in that particular and we can access them. sample, more cDNA will bind to their location and make that But this techniques does not allow us to look at location brighter. If the gene is not expressed a lot, less will bind to one specific gene. All we can say is that it is its corresponding spot --> spot will be darker. semi-quantitative but we then need to confirm it with more specific tools People have come up with search engine. If you put gene This is the same as previous experiment, just shown in graphic manner name, it will tell you how that gene scored in diff microarray -these are very good ways to get idea of how/where gene is experiments. Slide shows search for JAG expressed. But you need more specific methods (see next slide Datamining – eNortherns (U of T) for comparison of results Datamining – eFP browser (U of T) for comparison of results across microarray databases across microarray dytabases ➢ Nicholas J. Provart 1133 ➢ eFP Tool fr BAR at UofT; Dataset = AtGenExpress extended ti14ue series these numbers indicate relative expression level. Red= expressed at high levels at that developmental time (in this particular microarray, they took cDNA from diff times during development) Gross-tissue based methods: There are many ways to get more specific data about gene ex
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