Chapter 10 Analyzing Genes and Genomes

Chapter 10 Analyzing Genes and Genomes Copyright © Garland Science 2010 These slides were made to provide direction when reading Chapter 10 in Esse...
Author: Megan Perkins
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Chapter 10 Analyzing Genes and Genomes

Copyright © Garland Science 2010

These slides were made to provide direction when reading Chapter 10 in Essential Cell Biology—please read the sections corresponding to the figures in this ppt so that you thoroughly understand the concepts. I would like you to be very familiar with the techniques in this ppt, particularly the techniques that are underlined. We’ll spend time in class next week to answer any questions you might have. --Barb

Restriction Enzymes (Examples on next slide)



Cut DNA at defined sites (recognition site has to be exact)



Isolated from bacteria  Restriction enzymes (REs) are named after the bacterium from which they were isolated  Ex: EcoRI is from E. coli, strain R, and the 1st enzyme isolated from that strain; Hind III is from Haemophilus influenza, strain d, and the 3rd enzyme isolated from that strain



Bacteria produce these as ‘innate immune’ molecules—to cut any DNA from foreign invaders (note: bacteria that produce a given RE cannot have the recognition site in their own genomes)



We can buy isolated REs and use them to cut DNA of ANY species that has a sequence that is recognized. We call this, “restriction digestion.”



If a recognition sequence is 4 nts long, the likelihood of it appearing in any DNA (assuming random sequence) is 4x4x4x4=256 nts, and a 6 nt long recognition sequence is likely to occur once every 4x4x4x4x4x4= every 4096 nts

Figure 10-2 Essential Cell Biology (© Garland Science 2010)

Ligation • After DNA is cut with an RE, it often has ‘overhanging’ ends—any DNA (from ANY species) cut with the same RE will have the same overhanging ends. • If the DNA is mixed, the overhanging ends will be complementary and will hydrogen bond with any fragment of DNA with a complementary end. • If the enzyme DNA ligase is added to the mixture, the nicks in the DNA backbone can be sealed. In this way, 2 fragments of DNA, potentially from different species, can be ‘glued’ together to make a “recombinant DNA” molecule. • See the example on the next slide.

RESTRICTION DIGESTION WITH EcoRI

Ligation Reaction

Figure 10-6a Essential Cell Biology (© Garland Science 2010)

If we cut DNA with an RE, we can separate the fragments by gel electrophoresis.

mRNA

These figures to the left show separation of mRNA, but any nucleic acid can be separated in this manner, including DNA

Figure 10-3 Essential Cell Biology (© Garland Science 2010)

‘Northern blotting’ is a technique used to determine whether a specific RNA sequence is present in a given sample (tissue, cell, organ, etc.) • RNAs are isolated, separated by electrophoresis (by size), and transferred to a solid support (nitrocellulose) • The nitrocellulose ‘copy’ of the gel is incubated with a labeled (radioactive or color) “probe”; a “probe” is a DNA sequence that is complementary to the RNA • The labeled DNA probe binds to its complementary RNA (assuming it’s there) and because it is labeled, identifies whether your RNA of interest is present in the sample

Figure 10-5 (part 1 of 5) Essential Cell Biology (© Garland Science 2010)

Figure 10-5 (part 2 of 5) Essential Cell Biology (© Garland Science 2010)

Figure 10-5 (part 3 of 5) Essential Cell Biology (© Garland Science 2010)

Figure 10-5 (part 4 of 5) Essential Cell Biology (© Garland Science 2010)

Figure 10-5 (part 5 of 5) Essential Cell Biology (© Garland Science 2010)

Recombinant DNA can be ‘Cloned’

• Molecular cloning occurs when a molecule of DNA is ‘amplified’ (make many copies) • If we make recombinant DNA by ligating a fragment of interest into a bacterial plasmid (a non-chromosomal circular DNA that are isolated from bacteria), we can re-insert the modified plasmid into bacteria.  Once in the bacteria, the recombinant plasmid will amplify with the bacteria as they live/grow, so we can obtain many copies of our DNA fragment of interest (if we re-isolate it from the bacteria.

• See the next slides

Figure 10-9 Essential Cell Biology (© Garland Science 2010)

Figure 10-10 Essential Cell Biology (© Garland Science 2010)

Reverse Transcription • This is something we (eukaryotic cells) don’t do—even prokaryotes don’t do this. BUT, some viruses are able to convert RNA  DNA via reverse transcription, using a reverse transcriptase enzyme. • We can use this enzyme in the lab to convert RNA back to DNA.  This is especially useful if we convert mRNA into DNA because we can obtain a DNA copy (called ‘cDNA’) of the mRNA— which represents only the exonic parts of a gene. •

See the next slide

Figure 10-13 Essential Cell Biology (© Garland Science 2010)

PCR and RT-PCR • After converting RNA  DNA via reverse transcription, we can get billions of copies of the DNA by subjecting it to polymerase chain reaction (PCR). • The coupling of reverse transcription with polymerase chain reaction is known as RT-PCR. • The end result is billions of copies of a cDNA—DNA copies of an mRNA. • See the next slides

PCR PCR is basically an in vitro DNA polymerase reaction. We have a template, a DNA polymerase enzyme, buffers, nucleotides, and two primers and we simply let the polymerase reaction proceed. There are a few things that are different about PCR and a regular polymerase reaction. Mostly, our in vitro tube does not have all of the other enzymes (e.g., helicase, initiator proteins) that are in a cell, so we have to force the DNA apart by heating it. In addition, we do the reaction multiple times in tandem. PCR is a three-step process that is repeated multiple times in tandem. The three steps are: 1.) heating the DNA to separate the two strands of the template, 2.) lowering the temperature so primers can bind, 3.) allowing the polymerase to copy the template. Note that we have to include primers because the polymerase enzyme can’t start polymerizing but needs to add nucleotides onto a primer. The primers used in PCR are DNA primers—not like the RNA primers that are used in vivo. And we nee to include TWO primers because we want to copy both strands. See the next slide.

Figure 10-15 Essential Cell Biology (© Garland Science 2010)

These three steps are repeated over and over again in tandem to generate numerous copies of the original DNA template. Because the heating step is repeated numerous times, we need a heat-stable polymerase enzyme—one that can withstand the repeated heating steps. The polymerase we use is isolated from a bacterium that lives in thermal hot springs: Thermus aquaticus. Thus, we call the heatstable DNA polymerase “Taq polymerase.”

This figure shows just three cycles. We generally do at least 30, giving us billions of copies of double stranded DNA—the DNA that exists between the two primers that we’ve used.

Figure 10-16 Essential Cell Biology (© Garland Science 2010)

Comparison of PCR to RTPCR

Figure 10-17 Essential Cell Biology (© Garland Science 2010)

Another use of RT-PCR It’s a way to detect retroviral infection easily by collecting just a little bit of blood from a potentially infected individual.

Figure 10-18 Essential Cell Biology (© Garland Science 2010)

A use of PCR Look at the size of a specific DNA area (called a DNA ‘locus’; different individuals can have alternative ‘polymorphisms’ in their DNA

Figure 10-19a Essential Cell Biology (© Garland Science 2010)

Same thing as the previous slide, but for several different gene loci—if many loci are included in a genetic comparison, we can potentially [if enough loci are analyzed] whether two DNA samples are from the same individual (e.g., if the blood at the crime matches YOUR blood!) Figure 10-19b Essential Cell Biology (© Garland Science 2010)

DNA Sequencing It’s often necessary to know a specific sequence of nucleotides— for example, if we need to make probes for northern blotting or primers for PCR, we need to know those sequences. The method used to determine a DNA’s sequence is the same that has been used for several decades; it was developed by a researcher names Sanger, so it is often referred to as ‘Sanger sequencing’. Because of the nucleotides that are used, it is also referred to as ‘dideoxy sequencing’. This technique is basically just a modified DNA polymerase reaction (like PCR), so we would perform an in vitro polymerase reaction, with added di-deoxy nucleotides. See the next several slides.

No 3’ --OH

Figure 10-20 Essential Cell Biology (© Garland Science 2010)

If we include an appropriate amount of di-deoxy nucleotides along with regular deoxynucleotides, when DNA polymerase is ‘reading’ a template DNA, sometimes a dideoxy nucleotide will be incorporated into the growing strand, but most often a regular nucleotide will be incorporated so the polymerase keeps going. If you have enough template in a reaction tube, you’ll end up with polymerase products that stop at each and every place on the template. Figure 10-21 Essential Cell Biology (© Garland Science 2010)

If each of the lanes represents a single reaction tube containing a template DNA + DNA polymerase + appropriate buffers + a primer (remember, DNA polymerase always needs a primer to start—in this case, we use only a single primer because we only want to ‘read’ a single strand of the DNA [because we can deduce the sequence of the other strand) + regular dNTPs (all 4 of them, at least one of which is radioactively labeled) + 1 di-deoxy nucleotide, this is what you get when you subject the products of Sanger sequencing to gel electrophoresis. In the image of a gel to the left, lane 1 is a reaction with di-deoxy A included; lane 2 with di-deoxy G included; lane 3 with di-deoxy C and lane 4 with dideoxy T. The gel is read from the bottom up (because the shorter fragments go further in the gel during electrophoresis, thus those at the bottom are closest to the primer you put in the tube). Thus the sequence of this product is 5’-CGCGGGTCAAGTGGTTGACCT…. Test yourself to read the next 10 nucleotides. Remember that what you are reading is the product of polymerization—the template is complementary to this product. Figure Q10-10 Essential Cell Biology (© Garland Science 2010)

More modern techniques help us avoid using radioactive nucleotides, and also help us automate the process. Instead of radioactively-labeling nucleotides, we label them with color; each regular nucleotide is tagged with a different color. As a polymerase product comes off the bottom of the gel, a spectrophotometer can detect what color it was and records that color (and thus the sequence) in a computer. You can come look at the computer print out several hours later, and you have your sequence! Figure 10-22 Essential Cell Biology (© Garland Science 2010)

Expression cloning This is the same thing as molecular cloning, except that the plasmid that you will use is a bit different. It includes a promoter so that the fragment of DNA you ligate into the plasmid can be transcribed and translated into protein. Of course to do this, your recombinant DNA plasmid must be introduced into a cell—either prokaryotic or eukaryotic (different introduction techniques can/are used for different cell types). The most difficult aspect of expression cloning is that the fragment of DNA you ligate must be in the correct reading frame to produce your protein of interest. See the next slide.

Figure 10-24 Essential Cell Biology (© Garland Science 2010)

DNA Microarrays DNA microarrays are used to assess the relative expression of thousands of genes simultaneously—relative expression means that two things are being compared relative to one another. One isolates mRNA from two different sources (the two to be compared—e.g., normal cells and cancer cells, or lung cells of a smoker versus lung cells of a non-smoker). The mRNAs are converted to cDNAs using reverse transcriptase, and the cDNAs are tagged with different color labels, usually red fluorescent tags on the mRNA from one source and green fluorescent tags on the mRNA from the other. The color-tagged mRNAs are mixed, and incubated with a slide to which thousands of DNA fragments are bound in a grid. Each of the fragments is from a known gene, and there is a ‘key’ that lets researchers know where each of the known genes is located on the slide.

The figure to the left is from your text book—it shows the first four steps that are shown in the figure on the previous slide (from another text book). After binding (hybridizing) the labeled cDNA to the slide and washing, theoretically the cDNAs will bind to their complementary DNA fragments on the slide and stick there. Because they are colored, we can detect where the cDNAs are bound.

Figure 10-33 (part 1 of 2) Essential Cell Biology (© Garland Science 2010)

The figure on the left is a ‘cartoon’ of the grid on the slide—each dot on the grid represents a place where known DNA has been ‘dotted’. Again, presumably any labeled cDNA that is complementary to one of those dots of DNA will bind there. The figure on the right shows a grid to which the red and green-labeled cDNAs have been bound. A computer would read this and be able to tell whether there was more red bound to a dot, more green bound to a dot, or equal amounts of red and green (detected here as yellow). Thus, if there was more ‘red’ cDNA, this meant that the source that gave rise to that cDNA was expressing that gene (the one in the dot) more than the ‘green’ source; alternatively, if a dot is more green than red, it means that the source of the green cDNA was expressing that DNA at a higher level than the red source. If they are expressed in both sources equally, the dot will be yellow.

The analogous figure from your text book—this represents the last two steps in the figure shown on the first ‘DNA microarray’ slide. Figure 10-33 (part 2 of 2) Essential Cell Biology (© Garland Science 2010)

Generating Transgenic Animals We might do this to produce a number of different transgenic organisms (shown on the next slide—know them!). In either case, one typical use is to determine what effect a gene has on a particular process (so you take it out and see what happens, or you over-produce it and see what happens). A similar process is done with plants to produce genetically modified foods. In that case, a foreign gene might be introduced to have the plant produce some pesticide, or to be resistant to some herbicide. In the next slide, the red gene represents a gene that is modified from the normal, whereas the green gene is normal. Thus, panel c is showing something that might be used to determine whether expression of the mutant gene might ‘over-ride’ the normal gene in a ‘dominant-negative’ manner (this is analogous to a dominant mutation in an organism— you may have learned this in genetics class.

Figure 10-35 Essential Cell Biology (© Garland Science 2010)

Generating Transgenic Animals This is obviously a very complex process, and requires lots of steps , and hope that you can derive the nuances of this process from reading your text book. I’m going to simply put the steps here to produce an animal (mouse) that does not make your protein of interest. This is called a ‘knock out’ animal, and Dr. Mario Cappechi from the University of Utah receive a Nobel Prize a few years ago for developing this procedure. 1. Clone your gene of interest into an expression plasmid. The whole gene with introns. 2. Mutate your gene such that the transcription start site (and maybe another 1-2 exons) is deleted. This mutated gene is still in the expression plasmid. Most people also add a ‘selectable marker’ into the plasmid so they can tell whether or not the plasmid is present. An example of the ‘selectable marker’ is a gene for antibiotic resistance. 3. Introduce the mutated/truncated gene into an embryonic stem (ES) cell from a brown mouse. If the ES cells are cultured in the presence of antibiotic, then only the ES cells that contain the plasmid will be able to grow. 4. You pick up these cells and make sure that they have your mutated gene in them. These are the steps shown in panel A on the next slide. 5. After ensuring that you have stem cells with your mutant gene, you need to get an actual mouse embryo. You do this by harvesting them from a white mouse. Because it’s an early embryo, we can add in extra cells and the embryo will develop normally—this is because none of the cells in the embryo yet ‘know’ what they’re meant to be. 6. The embryo of mixed white and brown mouse cells is now implanted into a ‘pseudopregant’ female mouse. This just means that she’s ramped up on hormones and will ‘accept’ the implanted embryo. This is mouse IVF. 7. The mouse delivers some baby mice. If you’re lucky, the mice will have some of their ‘parts’ that carry the modified/mutant gene and therefore don’t’ express your gene of interest.

Figure 10-36 (part 1 of 2) Essential Cell Biology (© Garland Science 2010)

But wait! Step 7 above says that we hope that the mouse babies will have some of their ‘parts’ with the mutant gene. Actually, the only ‘parts’ we care about are the gonads. That is because the cells in these mouse babies have 2 copies of your gene of interest, one that you hope is mutated (i.e., missing it’s transcription start site) and therefore not being expressed. If the gonads have this mutation, then when gametes (sex cells) are made, the two copies of the gene (one mutated and one normal) will be separated; some of the haploid gametes will have the mutant gene only. So, step 8 is to let these mouse babies grow up and then you mate them with other normal mice. If their gonads carried the mutation, then some of the babies resulting from that mating will be heterozygous for the mutant gene. They’ll still have the normal gene, but they will be entirely heterozygous, and carriers of the mutation/deletion. 9. Genotype the mouse babies to see who is heterozygous for the mutation/deletion. 10. Mate the heterozygous animals to (hopefully) produce mice homozygous for the deletion (assuming that the total deletion of the gene is not lethal). And yes, you’re mating siblings! It’s gross, but they’re mice! Figure 10-36 (part 2 of 2) Essential Cell Biology (© Garland Science 2010)

That’s it. What do you think? Pretty cool stuff, huh? I hope it all makes sense. Again, we’ll revisit anything you are unclear about when I return. Till then, I hope your test went well, don’t forget your writing assignment/book report, and have a great week! See you November 5th.