topic 22

Question 1

how would you determine the probability that any one random n-bp sequence is a recognition site for a restriction enzyme xxxxxxx?

Answer 1

the probability of finding the correct base at each position is 1 in 4. therefore the probability of finding the correct base at all n positions is 1 in 4^n.

for example, if it was 2 bp, it would be 1/16

Question 2

the restriction enzyme recognizes the sequence with n-bp with a sequence of xxxxxxxxx. if you digest the human genome (with a random number of base pairs) with this enzyme, about how many unique DNA fragments would you get?

Answer 2

as an approximation, assume that each of the four bases occurs equally often in the genome, and that the order of the bases is random.

the odds of any n-bp sequence being xxxxxxxxx is then 1 in 4^n. thus the human genome should have a restriction enzyme cut site every 4^n bases or so (on average), giving rise to about (bp of human genome/4^n) = ______ DNA fragments

Question 3

an EcoRI-digested insert is to be cloned into a plasmid vector. before the ligation reaction, the researcher treats the EcoRI-digested plasmid DNA with alkaline phosphatase to remove the phosphates at the two 5’ ends. why treat the plasmid with phosphatase?

Answer 3

the researcher wants to prevent the two compatible sticky ends of the plasmid DNA from being ligated together without the insert. DNA ligase requires 5’ phosphate groups to join two strands together. note that the insert DNA will have 5’ phosphate groups, so DNA ligase will be able to join the insert to the plasmid in two places, which is enough for the plasmid + insert to remain circular. the two remaining nicks will be repaired after the plasmid + insert has been
taken up by the bacteria.

Question 4

what are the common method to check for gene expression? (2)

Answer 4

quantitative real time PCR (QRTPCR) and northern blotting.

Question 5

what is blotting? differentiate between northern and southern blotting

Answer 5

blotting refers to separating DNA, RNA, or proteins by size, then transferring them onto a membrane, probing for species of interest, and visualizing them. northern blotting detects RNA and southern blotting detects DNA. both use a DNA probe.

Question 6

describe the steps in the process of northern blotting

Answer 6

1. isolate RNA
2. separate RNA by size using gel electrophoresis. all RNA is negatively charged because of its phosphate backbone. one can denature RNA by running the gel at a warm temperature. people usually use agarose gel, where they put RNA samples individual welds at one end of the gel. then, cover the gel with a buffer and apply an electric current. negatively charged RNA will move through the gel, away from the negative electrode, towards the positive electrode. smaller pieces of RNA move faster than larger pieces due to the pores present in the polymer matrix.
3. transfer RNA to nylon/nitrocellulose membrane. usually one would use dye to the RNA but since it’s being transferred to a membrane, there’s no need. lay the membrane flat on top of the gel. then, put the gel and membrane on a sponge sitting in a salt solution and stick some dry paper towels on top (of the membrane). the salt solution will move up into the paper towel by capillary action and the RNA will move along with it, out of the gel and onto the membrane. after RNA has been transferred to the membrane, it’s no longer kept under denaturing conditions.
4. probe membrane for gene of interest. synthesize an oligonucleotide probe that’s complementary to part of the mRNA of interest and add a radioactive phosphate group to the 5’ end of the probe. incubate the membrane which has all of the RNA on it with a probe under conditions that will allow specific annealing of the probe to its complementary sequence. then, wash away the unbound probe and detect radioactivity on the membrane.

Question 7

describe Tm (melting temperature) and what factors determine it (7)

Answer 7

melting temperature (Tm) is the temperature at which 50% of a specific double-stranded DNA has become single-stranded (denatured). Tm is determined by many factors:

1. GC content: GC (+4°C) bonds are more stable and take a higher temperature to denature. they are stronger than AT (+2°C) because they have 3 bonds when AT has 2.
2. length: longer double helices melt at higher temperatures.
3. degree of complementarity: imperfect matches will have lower Tm.
4. salt concentration: counter-ions neutralize the negative charge of the phosphate backbone and reduce repulsion between strands. higher salt concentration = higher Tm
5. organic solvent concentration: they increase the hydrophobicity of the solvent and reduce base stacking interactions. a higher organic solvent concentration lowers the Tm
6. hydrogen bonding compounds: they weaken hydrogen bonding between bases. more hydrogen bonding compounds = lower Tm
7. pH: extremes pH changes the protonation states of the bases = lowering the Tm.

Question 8

describe the steps in the process of making a cDNA (4)

Answer 8

to make a cDNA copy of mRNA, an enzyme called reverse transcriptase is used. this enzyme is found in RNA viruses such as the coronavirus. this enzyme synthesizes a DNA strand using an RNA template, which can’t be done by DNA polymerase. note that cDNA contains no introns, unlike genomic DNA.

1. start with an mRNA prep.
2. synthesize cDNA using reverse transcriptase and a poly(T) primer. poly(T) nucleotides are used as a primer because they are complementary to the poly(A) tail. we end up with a DNA/RNA hybrid.
3. partly degrade the mRNA. to get a double stranded DNA started, the mRNA strand needs to be replaced with DNA. add an RNase enzyme to degrade the original mRNA. RNase H specifically degrades RNA that’s bound to a complementary DNA strand. it will degrade the RNA in random spots. as this happens, there will be parietal segments of RNA bound to DNA. these RNA fragments can act as primers for the DNA polymerase.
4. synthesize the 2nd DNA strand using DNA polymerase and ligase. the polymerase will use the partially digested mRNA as the 2nd strand. eventually, all RNA will be degraded and DNA ligase will join together any nicks in the newly synthesized DNA strand. the resulting double stranded cDNA represents all mRNA in the starting tissue.

Question 9

describe PCR

Answer 9

PCR is a method to amplify DNA. it’s a technique that allows one to make many copies of very small amounts of starting DNA.

the starting material can be cDNA or genomic DNA. the darker region on the DNA is the target region to be amplified. for cDNA amplification, the target region would be the entire cDNA molecule. the reaction mixture also contains 2 primers which are complementary to the template strands at either end of the region to be amplified. for PCR, must include a thermostable (able to retain its function even when heated to high temperatures) DNA polymerase, such as Taq, to synthesize new DNA. must also include dNTP( a substrate for the DNA polymerase) and a buffer (to provide conditions under which polymerase will be active).

to perform a PCR, must change the temperature of the solution in 3 stages and then repeat these changes for many cycles.

Question 10

describe the steps in the PCR process

Answer 10

1. raise the temperature very high (approx 95°C) which should cause all of the DNA in a solution to denature or separate into 2 strands, this is why a thermostable polymerase is needed
2. cool the DNA to a moderate temperature (approx 50-60°C) that will allow the primers to anneal specifically to the complementary sequences of their respective template strands.
3. raise the temperature to an optimal level for polymerase activity (approx 72°C). during this stage, the DNA polymerase will add nucleotides to the 3’ end of both primers, resulting in duplication the DNA and region to be amplified.

double stranded DNA consisting of only the target region to be amplified appears in the 3rd cycle of PCR. starting in cycle 3, the precise target region will grow in abundance.

Question 11

if one analyzes the PCR product by gel electrophoresis, what would they see?

Answer 11

they would see a single band representing the cDNA they wanted. the band can be take out of the gel and DNA can be extracted, giving a pure sample of the cDNA. this sample can be used in the next step of cloning.

Question 12

list applications of PCR

Answer 12

quantitation of target DNA using fluorescent dyes

DNA fingerprinting

site-directed mutagenesis

identifying virus or bacterial DNA in medical samples

diagnosis of genetic diseases

Question 13

describe DNA fingerprinting

Answer 13

an amplification of many genomic repeat sequences and compares samples among people. it relies on the fact that we have repetitive regions in our DNA that contain many repeat sequences. the number of times the short sequences are repeated varies from person to person.

Question 14

describe quantitation of target DNA using fluorescent dyes

Answer 14

can include a molecule in the reaction that becomes fluorescent in the presence of double stranded DNA. so, as a particular target sequence is amplified, the fluorescent signal becomes stronger. the fluorescence can be measured as the PCR proceeds to estimate the amount of PCR product made which should correlate with the amount of template made in the original sample. this is another way, besides northern blotting and microarray, to quantify the amount of specific mRNA expressed in a tissue of interest

Question 15

describe site directed mutagenesis

Answer 15

the mismatch in a primer is amplified. its used to introduce desired changes in a DNA sequence. design a primer that isn’t completely complimentary to the template sequence (contains 1 or more mismatches). as long as the 3’ end of the primer anneals to the template and is used by the DNA polymerase, it’ll generate a new DNA with that primer. that DNA will become the template in subsequent steps during which the mismatched regions will be copies into newly synthesized strands.

Question 16

describe plasmids

Answer 16

plasmids are relatively small pieces of DNA found in microorganisms that aren’t part of the organism’s genome, meaning they replicate independently of the chromosome. they are useful in introducing foreign genes. bacterial plasmids are usually circular, double stranded, and less than a thousand base pairs in size.

Question 17

what must a plasmid contain? why? (3)

Answer 17

plasmids must contain a replication original so the plasmid can be copied and spread to both daughter cells when the cell divides.

it’s very helpful to have a selectable marker on a plasmid, which is a gene that allows you to distinguish between cells that have plasmids and cells that don’t. often, the selectable marker confers resistance to an antibiotic. if the cells are grown in a media containing an antibiotic, cells with plasmids will survive and cells without plasmids will die.

many plasmids also contain multiple cloning regions which is selection of DNA that contains recognition sequences for many restriction endonucleases (RE) more specifically type II restriction enzymes.

Question 18

what is the purpose of a restriction endonuclease? how does DNA ligase “clean up” after it?

Answer 18

RE allows us to cut (double stranded breaks) and paste DNA fragments together with great precision. RE can recognize the sequence specific motifs and double stranded DNA. they are usually 4-8 base pairs long. they can generate “sticky ends” (contains overhangs) or “blunt ends”.

DNA ligase can join compatible sticky ends (is a phosphate is present on the 5’ ends) and blunt ends (with lower efficiency) respectively.

Question 19

how is a recombinant DNA formed?

Answer 19

the multiple cloning region has many RE recognition sites that allow us to cut open the circular DNA and paste in foreign DNA to create compatible ends.

incubate the fragments together to allow the ends to each other.

adding DNA ligase results in the sugar phosphate backbone being repaired. the DNA ligase requires a 5’ phosphate to be present in order to join the 2 strands together.

results in a recombinant DNA: an intact circular DNA that contains the desired insert.

Question 20

how is a plasmid introduced to a bacteria? what are the types? (2)

Answer 20

the process by which a plasmid is introduced into bacteria is called transformation. there are 2 main ways to transform bacteria.

1. electroporation: plasmid and bacteria are mixed under certain conditions. the cells are then exposed to an electric current, causing some cells to uptake the plasmid.
2. bacteria treated with chemicals to make it more competent (meaning the cells are capable of uptaking foreign DNA). mixing the cells and plasmids together and then quickly warming up the cells in a heat shock induces some cells to take up the plasmid.

in both methods, for the plasmid to remain stable in the bacterium, it must be circular. linear DNA doesn’t survive well in bacteria.

Question 21

what is the most stable shape a plasmid could have in bacteria?

Answer 21

for the plasmid to remain stable in the bacterium, it must be circular. linear DNA doesn’t survive well in bacteria.

Question 22

describe what happens after transformation

Answer 22

after transformation, incubate cells in agar plate containing antibiotics that corresponds to the selectable marker on the plasmids.

cells that contain the plasmid will be resistant and survive and grow on the plate. every colony on the plate is resistant to the antibiotics but guarantee that the plasmid contains the insert (cDNA) you want - could’ve been ligated without the insert.

therefore, the colonies must be tested to find the ones that contain the recombinant plasmid we tried to create.

Question 23

what are the methods you can use to check if the correct DNA has been inserted?

Answer 23

1. restriction mapping, where you cut the plasmid with restriction enzymes, then run the products on a gel to check the sizes of the fragments. since we know the sequence of the plasmid, we can predict how big the fragment should be and can tell whether an insert is about the right size that has been added to the plasmid.
2. do a PCR using primers that flank the predicted insert. if you see a plasmid of appropriate size, that means that your plasmid contains the insert.
3. denature the plasmid and incubate it with an oligonucleotide specific to the inset sequence. this could be done by southern blotting. if the probe binds to the plasmid, that suggests the insert is present.

out of these 3 methods, none guarantee that insert was incorporated into the plasmid in the way we wanted it to without any errors, it could have been a mutation. so, the most informative way to check for the correct incorporation of the insert into the plasmid is to determine the DNA sequence of the plasmid.

Question 24

describe the relationship between the degree of fluorescence and DNA fragment’s mass

Answer 24

the degree of fluorescence is proportional to the amount of bound dye, which is proportional to the mass of the DNA fragment.

Question 25

how would one check to see if the inserted DNA was done so correctly?

Answer 25

the most informative way to check for the correct incorporation of the insert into the plasmid is to determine the DNA sequence of the plasmid. that way you can see exactly what happened during the digestion, ligation, and transformative step.

to obtain the DNA sequence of the plasmid, we use the Sanger DNA sequencing method

Question 26

how would one analyze the results from the step where we checked to see if the inserted DNA was done so correctly through the sanger sequencing method?

Answer 26

there will be millions of copies of templates in each reaction mixture. each time a new strand is made, synthesis will stop at random. so, you will end up with a series of newly synthesized fragments, each stopping a different point in the sequence, and each fluorescently tagged according to the last base that was added.

to analyze, you denature the strands and separate them by capillary gel electrophoresis. the small strands move more quickly through the capillary than the larger strands. monitor the bottom of capillary for fluorescence, to read the complement to the DNA strand you want to sequence. each peak represents the signal from a strand with a given length with the colour of fluorescence indicating the base present at that position of the strand.

the computer can convert the peaks into a sequence. sanger method is useful when you have a relatively small number of samples to analyze.

Question 27

describe the sanger sequencing method

Answer 27

Sanger DNA sequencing method aka dideoxy sequencing aka chain termination sequencing. in this method, we set up a DNA synthesis reaction with a primer that anneals to the DNA that’s being sequenced, a DNA polymerase, dNTPs, and a small amount of ddNTPs (which are similar to dNTPs except they lack a hydroxyl at both the 2’ and 3’ carbon of the sugar). if there’s ddNTPs incorporated into the growing chain, DNA synthesis will stop at that point because there’s no hydroxyl onto which the next nucleotide can be added to. that’s why ddNTPs are also called “chain terminators”. you include a bit of all 4 ddNTPs into the reaction mixture so DNA synthesis can stop at any base on the template. each type of ddNTPs has a differently coloured fluorescent label on it which helps one to determine which base was incorporated at the terminating position.

unlike PCR, where there’s 2 primers, only 1 primer is used here.

Question 28

describe the steps in the process of purifying a protein

Answer 28

1. create expression plasmid
2. transform cells with plasmid (first 2 steps already done!)
3. grow cell culture, inducing overexpression: to overexpress a protein in E. coli, you insert the gene of interest into an expression plasmid after a regulated promoter.
4. lyse cells (break cells open)
5. remove cellular debris (with centrifugation to remove remnants of cell wall/membrane)
6. column control chromatography (affinity to a specific matrix). some people will engineer affinity tag into their protein of interest
7. check purity: 1st, equilibrate material that binds to the affinity tag to make sure you have everything in the same buffer. then, load the sample onto the column. only the protein of interest should have the tag and bind to the column. unbound protein will flow through. to release the bound protein of interest, add a competitive binder that displaces your protein from the column. your protein should now be halfway pure.

Question 29

what type of chromatography would be used for histidine purification or a poly-histag on your protein?

Answer 29

for histidine purification or a poly-histag on your protein, you can use nickel affinity chromatography.

Question 30

describe the structures in a protein expression vector (6)

Answer 30

a vector is used to move some DNA of interest into a desired location.

1. plasmids must have an origin of replication so the cell can make more copies.
2. the plasmid should also have a promoter that the bacterial RNA polymerase can recognize. ideally, this promoter would contain regulatory sequences that allow transcription of the gene to be turned on at a specific point in the growth of the bacterial culture. need to make sure the mRNA has a ribosome binding site so the mRNA can be translated.
3. to simplify purification, you want a protein affinity tag included in the gene. the tag is usually put at the 5’ end or 3’ of the gene so that the tag will be found at the N-terminus or C-terminus of the polypeptide.
4. often, you include a sequence (coding sequence for tag removal) that is the target for a specific protease, such that the affinity tag can be removed from the protein product. if the affinity tag is small enough, it doesn’t matter if it stays on.
5. you should include a transcription terminator so the RNA polymerase knowns when to stop making mRNA.
6. plasmids should also have a selectable marker so you can identify cells that contain the plasmid.

Question 31

describe transgenic organisms

Answer 31

transgenic (genetically modified) organisms are made to investigate the function of a protein in the context of an organism.

they have genomes that have been permanently altered through genetic engineering, this is different from adding a plasmid to an organism because the plasmid is not part of the genome and can be lost.

manipulating a gene in an organism’s gene is a good way to gain information about what the gene product is doing in an organism.

Question 32

list and describe the types of transgenic changes (3)

Answer 32

gene replacement: with a modified version

gene knockout: eliminate

gene addition: add. usually results in high expression

Question 33

describe CRISPR-Cas9 and how it works

Answer 33

(clustered regularly interspaced short palindromic repeats) CRISPR-Cas9 is an endonuclease that creates double strand breaks in DNA. unlike restriction endonuclease which cuts DNA at a defined unchanged target sequence, Cas9 is directed to cut specific sites on a DNA by a guide RNA that binds to the enzyme. without the guide RNA, Cas9 will not cut the DNA. the guide RNA is about 30 nucleotides long and 20 of those specify the DNA site Cas9 will cut. after Cas9 has made a double stranded break in the genome, the organism’s double strand break repair machinery will try to fix the break.

Question 34

after Cas9 has made a double stranded break in the genome, how will the organism’s double strand break repair machinery try to fix the break?

Answer 34

if they do so by non-homologous end-joining. then it’s likely the bases will be lost at the repair site. if the site is towardmicros the beginning of the gene, it’s likely that the gene will be inactivated due to a gene knockout.

otherwise, the organism mist repair the double stranded break by homologous recombination. if the researched has provided replacement DNA, there is a chance that the organism will copy that during its repair of the double strand break, in which case, the altered version of the gene of interest could be created.

alternatively, an entire gene could be added at the site of repair. homozygous knockout is much more dangerous than heterozygous knockout.

Question 35

what purpose does oligonucleotides serve?

Answer 35

oligonucleotides can be used as s hybridization probes and as primers for DNA sequencing, PCR and mutagenesis