Unit 9 Flashcards
Describe the five general procedures in DNA cloning
- Obtaining the DNA segment to be cloned
- Selecting a small molecule of DNA capable of autonomous replication
- Joining two fragments covalently (through DNA ligase)
- Moving recombinant DNA from the test tube to a host organism
- Selecting or identifying host cells that contain recombinant DNA
Thousands of restriction endonucleases have been discovered. Are restriction enzymes made by eukaryotes or prokaryotes?
Prokaryotes
What is the biological function of restriction endonucleases in vivo?
To cut out foreign DNA
Discuss the restriction-modification system
- In a host cell’s DNA, the sequence that would be recognized by one of its own restriction endonucleases is protected from digestion by methylation of the DNA, catalyzed by a specific DNA methylase
- The restriction endonuclease and DNA methylase are referred to as teh restriction-modification system
Select a restriction endonuclease that generates sticky ends and one that generates blunt ends
Sticky: BamHI, EcoRI
Blunt: HaeII, PvuII
Sticky ends
Some restriction endonucleases make staggered cuts on the two DNA strands, leaving two to 4 nucleotides of one strand unpaired at each resulting end. There unpaired strands are referred to as sticky ends because they can base-pair with each other or with complementary sticky ends of other DNA fragments
Blunt ends
Some restriction endonucleases cleave both strands of DNA straight across as opposing phosphodiester bonds, leaving no unpaired bases on the ends, often called blunt ends
How are the cloning vector and desired DNA combined?
When two DNA fragments generated by the same restriction endonyclease are mixed together, the complementary ends pair and form hydrogen bonds (a process called annealing), DNA ligase then forms phosphodiester bonds that covalently join the fragments, and the restriction endonuclease cleavage site is restored
What is another name for small molecules that contain DNA capable of self replicating?
Cloning vectors
What are the cloning vectors
- Plasmids
- Viruses
Plasmid
A circular DNA that replicated separately from the host chromosome
Transformation
- In the laboratory, small plasmids can be introduced into bacterial cells by a process called transformation
1) plasmid and bacteria cell are incubated together at 0ºC in NaCl solution
2) Heat shock for 37-43ºC
3) Cells may take up plasmid DNA
Why do plasmids always contain each of the following regions:
the selectable marker gene
A selectable marker gene either permits the growth of a cell (positive selection) or kills the cell (negative selection) so you can identify the population of your bacteria that successfully took up the plasmid and recombination DNA
Why do plasmids always contain each of the following regions:
the origin of replication
This sequence is required to propagate the plasmid. It’s wehre replication starts
Why do plasmids always contain each of the following regions:
unique recognition sequences for restricton endonucleases
Several unique recognition sequences are targets for restriction endonucleases, providing sites where the plasmid can be cut to insert foreign DNA
How plasmids are used to clone DNA
- Plasmids are first cleaved by a restriction endonuclease
- Then, the foreign DNA is cleaved by the same endonuclease will combine with the plasmid
- DNA ligase seals them up
- The cells of interests are transformed then grown on agar plates with compounds that hep the selectivity of the bacteria
What are the two sources of error with doing uptake of plasmids?
- The plasmid did not get the DNA
- The host cell did not pick up a plasmid with recombinant DNA or a plasmid at all
On page 14 make sure you can explain this process
What kind of vector will you use if your goal is to express a eukaryotic gene?
Expression vector
What are the components of an expression vector (make sure you can draw out this diagram)
- Ori
- Gene encoding for repressor
- Promoter
- Operator
- Ribosome binding site
- Multiple cloning sites (cDNA enters)
- Transcription termination sequence
- Selectable gene markers
Promoter
Allows efficient transcription of the inserted gene
Operator
Permits regulation by a repressor that binds to it
Ribosome binding site
Provides sequence signals for the efficient translation of the mRNA derived from the gene
Multiple cloning sites
The gene to be expressed (cDNA) is inserted into one of these restriction sites, near the promoter, with the end of teh the gene encoding the amino terminus positioned the closest to the promoter
Transcription termination sequence
Improves the amount and stability of the mRNA produced
Gene encoding repressor
Creates repressor that binds to the operator and regulates the transcription of the gene
*there is also ori but we’ve already discussed what that is and its importance
Why do we use cDNA instead of just using the DNA from the eukaryote?
The reason why is because prokaryotes do not have the machinery to remove introns. If we were to transcribe DNA directly, it would have introns included which we don’t want
Therefore, what we must to is take our DNA –> primary mRNA –> mature mRNA and use a reverse transcriptase to turn that into cDNA. Now this version of DNA does not have any introns and the promoter can activate the mRNA of this DNA
Why is it necessary to have a promoter in the expression vector
Because once you have a promoter, you will be able to express the DNA to make RNA
With very strong promoters, expression vectors sometimes recruit many of the cell’s transcription and translation enzymes to “over-express” the cloned gene
Cloned genes are so efficiently expressed that their protein product represent 10% or more of cellular proteins. At these concentrations, some foreign proteins can kill the host, so expression of cloned gene mut be limited
Why does site-directed mutagenesis matter?
Sometimes we want to study proteins and changes accompanied with that. To do so, we must alter the gene. SIte directed mutagenesis changed the DNA (gene) so it may encode a different protein product
What are the two ways to have site-directed mutagenesis
- Synthetic Insert
- Oligonucleotide-directed mutagenesis
Define site-directed mutagenesis
A set of methods to create specific alterations in the sequence of a gene
Synthetic Insertion
- When you have a plasmid that you cut through restriction endonucleases and insert a desired gene that was identical to what was cut out at the plasmid except for it has maybe one altered nucleotide (making it an mutated)
Oligonucleotide Directed Mutagenesis
- You have specific primers that are complementary to the target region you want to change except there is one mutation in the primer (the mutation you want to observe)
- Even though it isn’t 100% complement, it’s close enough to have the primer bind to the gene sequence
- Then, you have a DNA polymerase come in and use the primer to replicate the rest of the DNA, using the original strand as a template
- Now you have a new DNA strand with you said mutation included
Steps for Oligonucleotide Directed Mutagenesis
- Denature plasmid and anneal oligonucleotide primers with mutation
- Use DNA Polymerase to extend and incorporate the mutagenic primers
- Digest non mutated parental DNA template with methylation-specific nuclease, and anneal newly synthesized strands
- Transform dsDNA into cells. Cell repairs nicks in mutated plasmid
How to synthesize single stranded DNA primers
- Attach nucleoside to silica support. The nucleotide has a DMT protecting it’s 5’ HYDROXYL GROUP
- Then, remove the DMT protection to add your next nucleotide
- Let’s say you wanted to add cytosine, you would then flood the nucleotide with dCTP’s that also have a DMT blocking the 5’ hydroxyl site so you don’t have addition of more than one nucleotide at a time
- Repeat the process until you have your desired primer
Suppose you want to use oligonucleotide-directed mutagenesis to make a mutant gene taht encodes a protein containing phenylalanine rather than tyrosine. Given the following DNA and protein sequence, write the sequence of primers you would design to make the mutant protein Coding strand is show 5’ to 3’
- First see the amino acid sequences that you are trying to change
- Figure out what the mRNA code is for the original amino acid
- Figure out what the mRNA code would have to be to make one amino acid change
- Go backwards and find what DNA would encode that mRNA (through base pairing) –> this is one primer
- Do the complementary of the first primer to give you the second primer
!!! be very careful. Sometimes they may give you the coding strand. In this case, whatever your mRNA is with the mutation change is the exact same as your coding strand, except your U’s are swapped for T’s!
Everytime you see coding strand, what does that mean??
It’s the exact same as the mRNA but you are swapping out the U’s for T’s
Describe a procedure used to tag proteins thereby facilitating their purification using affinity chromatography techniques
- Tag sequences can be added to genes such that the result proteins have tags at their amino or carboxyl terminus
GST Tag
- A column is filled with a porous matrix consisting of the ligand (glutathione) immobilized on microscopic beads of stable polymer such as cross-linked agarose
- As the crude extract moves through the matrix, the fusion protein becomes immobilized by binding the glutathione
- The interaction between GST and glutathione is tight but noncovalent, allowing the fusion protein to be gently eluted from the column with a solution containing either a high concentration of salts or free glutathione
What is a His tag and what is it used for?
- Simple sequence of 6 or more His residues
- They bind tightly to Nickel ions
- This can be used in chromatography where a protein with a His tag can bind to immobilized bickel ions and be removed from the column
What is the only thing you have to know for PCR?
Have to know the sequences of the portion you want to replicate (especially the endings) so you can create the correct primer
What is needed in order to amplify a segment of DNA using PCR
- DNA sample containing the segment to be amplified
- Pair of synthetic oligonucleotide primers
- dNTPs
- Taq DNA Polymerase
Discuss the selection of primers and the three repeated steps in the process: Heating, Cooling, and Replication
Primers are selected based on the ends of the target sequence you want to amplify
1. Reaction mixture is heated briefly to denature the DNA, separating the strands
2. The mixture is cooled so that the added primers will anneal to the DNA
3. The primed segment is replicated selectively by the DNA polymerase
This cycle is repeated 25-30 times
Draw out the process for PCR *
For each copy of the target sequence that you started with, how many copies do you expect after cycle 10?
2 ^ (number of cycles )
2^10 = 1024 copies
A normal enzyme is denatured above 50-60ºC, but in each cycle of PCR the mixture is heated above 90ºC. Explain why use of DNA polymerase from the bacterium Thermus aquaticus made a big improvement in this procedure
Taq polymerase is used because it is stable at high temperatures so that when the DNA is denatured and the solution is heated, the Taq polymerase won’t denature and lose its structure/function
Among its many other uses, PCR is finding important applications in early clinical diagnosis of disease. An example of testing for the presence of viruses to which antibodies do not appear until a long time after infection. Describe how PCR could be used to detect HIV infection at an early stage, including what information about HIV would be needed to begin with
- Perform PCR on cells with primers that are designed to amplify the viral HIV DNA that is integrated into the host genome
- You would have to know the sequence of the HIV gene to make a complementary primer so you know where it should start.
Do Problem 5
Typically, we would want to include the primer at the very beginning of our target sequence. However, since huntington’s is repeated sequences, we would have to design a primer that can bind to a more unique sequence that isn’t the same. Or else, there would be no specificity for where the primer should bind
Do Problem 13
We know that to design primers, we want one primer on one strand of DNA (starting with the desired portion) and another primer on the other end
- We know that DNA strands are antiparallel so it helps to draw a visualization of antiparallel DNA strands getting primers added to them
- Note that when adding the primer for the 5’ to 3’ strand (remember 3’ strand is the bottom strand), the primer is at the end of the 3’ region. Therefore, you primer would have the last 20 nucleotides in at the end of the 3’ region.
- Now we have to write the complementary of this DNA strand since they only gave us one strand. We know that the first few nucleotides here, closer to the 5’ end, will be the nucleotides we use
Show how primers can be used to create restriction endonuclease cleavage sites on your PCR product to facilitate cloning. In this example, 5’ BamHI site is added to the gene
What if you wanted to use PCR for cloning?
- Primers can have restriction endonuclease cleavage sites added on to them that are not complementary to the template strand (therefore, they’ll be hanging off)
- When the PCR produces the two dominant strands, those unique recognition sites will be present because they were included in the primer even though they didn’t bind
- An endonuclease that is specific for that unique recognition site will come and cleave it
What does RT-PCR stand for and why is it used?
- Reverse Transcription Polymerase Chain Reaction
- It is used to amplify RNA sequences that may be of interest (can be used to detect sequences from living cells)
Describe the RT-PCR method, naming both enzymes involved
*Only the beginning of RT-PCR is a bit different because it uses an enzyme called a reverse transcriptase
1. Obtain RNA sequence and add oligonucleotide primer to it
2. Add reverse transcriptase to synthesize a cDNA strand paired with a RNA strand
3.Use heat to separate the strands
4. Add oligonucleotide primer to the DNA strand (RNA strand is now unused)
5. Use Taq DNA polymerase to synthesize the rest of the DNA strand
The rest is PCR again
Draw out how RT-PCR works
What does qPCR stand for and why is it used?
- Quantitative Polymerase Chain Reaction
- It’s used to quantify/estimate the amount of copies of a particular sequence in sample, or to detect sequences that are present in higher amounts
How does qPCR work?
- You have a DNA probe that is a hairpin. It is complementary to the target gene
- At one end of the DNA problem you have a fluorophore and you have a quenching molecule on the opposite site.
- As long as the fluorophore and quencher molecule are in close proximity to one another, the fluorophore cannot emit a signal
- However when the probe gets to the target DNA, it will open up to become the complement of the target DNA
- This separates the fluorophore from the quenching molecule, allowing for fluorescence
What is a CT?
The cycle number at which it took the sample to pass the threshold
What does a low CT value mean?
It means it passed the threshold quicker so it has more copies
What is a DNA library?
A collection of DNA clones, or usually gathered for purposes of gene or protein function
Name types of DNA libraries
- cDNA library
- Combinatorial gene or gene library
cDNA library
a collection of cloned DNA fragments derived from the complement mRNA being expressed
Combinatorial gene library or gene library
Focuses on sequence variants within one gene. For example, beginning iwth the cloned gene of enzyme X, a segment of the gene could be replaced with nearly identical fragments synthesized with a slight imprecision
Transcriptome
The entire complement of RNA transcripts present in a given cell or tissue under specific conditions
Describe how RNA seq is used to analyze the transcriptome in a cell
- RNA is first isolated from tissue or population of cells
- RNA is fragmented and converted into double stranded DNA using reverse transcriptase (because it would be too long to encode the entire transcriptome)
- DNA is subjected to deep DNA sequencing, which reveals both the RNAs that are present and the relative abundance of each (if more copies of one RNA are present, they will give rise to more DNA sequencing reads)
Fusion proteins
Ligating the genes for two domains (or proteins) to make a protein with both
List the fusion proteins
- Purification (like GST)
- Immunoprecipitation
- GFP
List all understanding protein methods
- Purification
- Immunoprecipitation
- GFP
- Immunofluorescence
- Yeast-2-Hybrid Analysis
Describe GFP and its variants
A useful marker usually fused to the target gene to generate a protein that is highly fluorescent (lights up when exposed to blue light)
Variants of GFP are now available in almost any color by the visible spectrum with other types of fluorescence
Discuss how fusion with the gene of GFP can be used to localize specific proteins within cells and organisms
GFP is fused to a protein, sites of fluorescence reveal location of proteins
Discuss how indirect immunofluorescence microscopy can be used to determine the subcellular localization of a specific protein
- Alternative for GFP if the protein is inactive or not expressed in sufficient levels
- The approach requires a fixed (dead cell) and the cell is fixed and permeabilized
- The protein of interest is sometimes expressed as a fusion protein with an epitope tag that is bound tightly to a commercial antibody (could or couldn’t have a fluorophore )
- A second antibody is added that can bind to the first open so it has a fluorochrome attacked
Compare and contrast GFP-tagging and indirect immunofluorescence by noting the advantages of each method
GFP can be done with live cells
GFP might not always have great expression
Immunofluorescence has to kill, fix, and permalize the cell
Can have nonspecific binding
Describe how fusions with epitope tags can be used to identify interacting proteins
- Fuse a gene encoding a protein with an epitope tag. Investigators can precipitate the protein product of the fusion gene by complexing it with the antibody that binds the epitope 9so it will form precipitate at the bottom)
- Other proteins that bind with the tagged protein will also precipitate with it
- You can then identify the associated (that were bound to the target protein) proteins through gel electrophoresis
What are three tags we’ve learned about?
His tags
GST tags
Epitope tags
Describe how the yeast two-hybrid analysis is used to identify interacting proteins
- Two different protein domains make up the protein Gal4p
- One domain is the DNA binding domain and the other domain is the activating domain.
- When these domains are together, transcription occurs; however, they are found on two different fusion proteins
- The two fusion proteins when they come together will allow for transcription.
- They will also transcribe some reporter gene that notifies scientist if the gene was transcribed or not (if the proteins interacted or not). Examples of reporters could be GFP, or some metabolic change
What does CRISPR stand for?
Clustered Regularly Interspaced Short Palindromic Repeats
How can CRISPR/Cas9 be used to “knock out” a gene. What components are needed for this gene knockout approach
- Cas9 endonuclease
- sgRNA
- target DNA
Show how CRISPR/Cas9 can be used to knockout a gene
The sgRNA (that already incorporate the necessary change) will go to the double stranded DNA. The Cas9 endonuclease will break both strands. sgRNA will include its complement in the breaks. Cas9 will break both strands again and a DNA ligase will combine them causing a K.O
DOUBLE STRANDED
How can CRISPR/Cas9 be used to make a specific change in a gene sequence
The sgRNA (that already incorporate the necessary change) will go to the double stranded DNA. The Cas9 endonuclease will break both strands. sgRNA will include its complement in the breaks. Cas9 will then break one strand. The recombinant DNA that has homologous ends to the surrounding sequences of the broken strand will fuse together.
What additional components are needed for this application of CRISPR that is not needed for knock outs
- Recombinant fragments
*cas9, sgRNA, target DNA
What are micro-RNAs
A class of small RNA molecules involved in gene silencing by inhibition translation and or promoting degradation of particular mRNAs
Explain how RNAi can be used as a gene silencing method
RNAi can degrade or stop translation depending on how well is base pairs with the complementary strand
Discuss the Sanger method for DNA sequencing
You will need:
- DNA polymerase
- dNTPs
- ddNTPs
- radioactive primer
- DNA template
*ddNTPs stop replication because they have an H instead of an hydroxyl group at the 3’ carbon
- You will have synthesis of the DNA strand as usual, adding complementary base pairs to the template strand.
- Depending on which ddNTP molecule you have, there is a change it can bind to its complement and end synthesis.
- It can end synthesis and create fragments at any place it has its complement and there’s an additional change that the ddNTP just doesn’t bind at all and you have synthesis of the whole strand.
- Therefore, you must have a reaction with each of the 4 ddNTPs separately
- At the end, run it in the electrophoresis. The shortest strands will move the furthest on the electrophoresis so that will be the nucleotide closest to the 5’
- You also must remember that the sequence that comes up on the electrophoresis is in the 5’ to 3’ direction, making it the synthesized/coding strand
- To find the template strand, you must find the complement of that
Draw the structure of ddATP. Why does it affect polymerization by DNA polymerase
By incorporating ddNTP, you are stopping polymerization becasue you no longer have a 3’ Oh group taht can attach
Dont forget that when you draw it, its a triphosphate
How many reactions are included in a typical DNA sequencing experiment
4
Describe the composition of each reaction
DNA polymerase
DNA template
ddNTPs
dNTPs
radioactive primer
Review the definition of “radioactive isotope” given in the glossary of the textbook. How is radioactivity incorporated into the sequences
- An isotopic form of an element with an unstable nucleus that stabilizes itself by emitting radiation
- Gel electrophoresis is ran and the gel is exposed to x-ray film. The radioactive primers emit radiation on the gel and the exposure appears as a dark band
Given the sequence and primer, write teh sequences that would be produced using the dideoxy analog of dATP. Do the same for the other three dideoxy analogs
- For these questions, ddATP will bind with T’s, ddTTP will bind with A, ddGTP will bind with C’s, adn ddCTP will bind with C’s
- Once they bind they stop polymerization and create a fragment
- Draw all the fragment possibilities and add an extra fragment possibility because all of the sequence can be formed and the ddNTP might not bind
The band seen on the gel electrophoresis differ in length by how many nucleotides?
There is a spacing of one nucleotide between all of the bands
Do problem 18
- So for questions like these, they gave you the sequence of DNA and the mixture 1. So we know that mixture 1 has ddTTP so wherever there is an A, it will be fragmented
- So label the gels and explain which one is which fragment
- Then for the next question, you can draw the fragments of C caused by ddGTP relative to the first mixture
- If you’re missing a nucleotide and you the the dideoxy nucleotide version of that, wherever the fist site for it to bind complementary to will be the one and only fragment. So for that example, ddTTP would bind to the first A and that would be the only fragment that would appear
- Lastly, if you don’t have any ddNTPs, you would just have DNA polymerization as normal, meaning you have one sequence at the very top because it is the longest and not fragmented
Steps of automated Sanger method
- Each ddNTP can be linked to a fluorescent molecular that gives the same color to all the fragments terminating in that nucleotide (so first the segments are fragmented and the fluorescent is added to the ddNTPs)
- Then they are denatured and separated in size in an electrophoretic gel in a capillary tube.
- The color associated with each bad is detected with a laser beam
Why is it possible with this method to have only a single reaction mixture?
It’s possible to have only a single reaction mixture because if you are giving each ddNTP a different fluorophore, you can distinguish tehm
After separating the fragments, how do you know which fragment stopped at each residue
Based on the fluorescence emitted at that base and specific time
Why does this method allow more sequence information to be derived from a single sequencing reaction?
DNA sequence is read by determining sequence of colors in peaks as they pass through the detector. This info is ged directly into a computer that determines the nucleotide sequence
Can handle a large number of samples in a short time
Describe next-generation reversible terminator sequencing
- Add blocked, fluorescently labeled nucleotides (add all your dNTPs and the correct one will bind complementary)
- A fluorescent color is observed and recorded
- Remove labels and blocking groups; wash; and add the next set of blocked nucleotides
- Fluorescence is observed and it will occur again
Why is next gen advantageous
- For big genomes
- Doesn’t need ddNTPs
- has blocking groups attached to fluorescently attached dNTPs
Explain how the short sequences generated using this method are assembled into contigs
Computerized alignments of overlapping fragment sequences allow computers to trace sequence through chromosome from one fragment to another, assembling the contigs
What does SMRT sequencing stand for
Single molecular real time sequencing
What is the advantage of SMRT sequencing over reversible terminator sequencing
- Most accurate form of sequencing
- Facilitate the detection of genomic alterations-deletions
Describe how SMRT sequencing works
In SMRT sequencing, template DNA is first prepared by fragmenting it into small pieces. These fragments are then ligated to specialized adapters that enable them to bind to the surface of the SMRT cell. Once attached, the DNA polymerase enzyme is immobilized within a zero-mode waveguide (ZMW) on the SMRT cell. As the template DNA passes through the ZMW pore, the DNA polymerase incorporates fluorescently labeled nucleotides during DNA synthesis. The emitted light is detected by a camera, allowing the sequence information to be determined in real-time.
I apologize for any confusion. In SMRT sequencing, the template DNA molecules are indeed fragmented into smaller pieces. However, during sequencing, each fragment is processed individually in the ZMW pore, resulting in the synthesis of a single complementary DNA strand for each fragment. So, while the original template DNA may be fragmented, the sequencing process itself generates a single contiguous sequence for each fragment.
How many protein coding genes in the human genome
20,000
What percentage of the human genome consist of genes
27.4
What percentage of the genome encodes proteins
1.5
What are transposons, what percentage of the genome do they represent
Segments of DNA, ranging from a few hundred to several thousand base pairs long, that can move from one location to another (2.9%)
Define SNP
Small nuclear polymorphisms
A genomic base-pair change that helps distinguish one species from another or one subset of individuals in a population
What kind of bond does DNA ligase make to seal strands of DNA?
Phosphodiester
