Term
| Define Result and Conclusion |
|
Definition
Result—product of an experiment or other—raw data—observations
Conclusion—results make up the conclusion, this is a general statement about data, interpret results |
|
|
Term
Gene cloning Step 1: ___A YGI from among thousands of genes
2. ___B YGI to bacterial (or phage--virus taht infects bacteria) plasmid DNA
3. ___c recombinant plasmid into bacterial (or phage) host by ______d
4.___E individual cells/clones
5. ____F sufficient amounts of YGI's DNA and/or protein for further analysis |
|
Definition
A. Isolate
B. Link
C. Introduce
D. Transformation
E. Separate
F. Make |
|
|
Term
| What results in Recombinant molecules? |
|
Definition
| Cloning DNA pieces (i.e. cutting from one source and pasting into another molecule) |
|
|
Term
| what does recombinant mean? |
|
Definition
| Non native--not originally part of the organism's DNA |
|
|
Term
| What makes a recombinant protein? |
|
Definition
| Expression of a foreign gene in bacteria. |
|
|
Term
|
Definition
| they cleave a piece of DNA internally, generating 2 pieces from one (2 strands) |
|
|
Term
|
Definition
| non specific at the end of a DNA molecule |
|
|
Term
| What did Ham Smith discover? |
|
Definition
| Specific restriction endonuclease (HindII) |
|
|
Term
| What organism is HindII come from? |
|
Definition
|
|
Term
| What does HindII recognize and CUT? |
|
Definition
5'GTPyPuAC3'
3'CAPuPyTG5' |
|
|
Term
| What are the Pyrimidines? Purines? |
|
Definition
CUT the PYE
Pure(ines) As Gold |
|
|
Term
| What does HindII Create (blunt or sticky ends)? |
|
Definition
| Blunt end b/c it cuts in the middle |
|
|
Term
|
Definition
|
|
Term
|
Definition
| Non uniform staggered cut. |
|
|
Term
| What RE can cause a Sticky end? |
|
Definition
|
|
Term
|
Definition
5'-G3' || 5'AATTC3'
3'CTTAA5' || 3'G5' |
|
|
Term
|
Definition
| one that cuts every 6 nucleotides |
|
|
Term
| How do we determine probability of cut? |
|
Definition
1/4 ^ x
x being the number of nucleotides per sequence |
|
|
Term
| What is a heteroschizomer? |
|
Definition
| Recognize the same sequence, but make different cuts |
|
|
Term
|
Definition
| Recognize the same sequence and make the same cut |
|
|
Term
| What is the origin of a restriction enzyme? |
|
Definition
| Produced by bacteria as a defense mechanism--cut up invading viral DNA |
|
|
Term
| Why don't RE cut host DNA? |
|
Definition
| RE are paired with methylase in "restriction-modification system" protecting it from digestion/cutting |
|
|
Term
| What is methylated in an organism? |
|
Definition
| The DNA from the organism not any other. |
|
|
Term
| What is hemimethylation of DNA? |
|
Definition
| only one strand is methylated, but still provides adequate protection from REs |
|
|
Term
| What are the 3 steps in inserting Recombinant DNA? )Refer to slide 11 |
|
Definition
1. Restriction enzyme cuts sugar-phosphate backbones
2. DNA fragment from another source is added. Base pairing of sticky ends produces various combinations
3. DNA ligase seals the strands |
|
|
Term
| Besides RE and ligase a simple gene cloning experiment needs |
|
Definition
|
|
Term
|
Definition
| A small circular piece of DNA that is independent of the host chromosomes. |
|
|
Term
| What does the plasmid need before replication can occur? |
|
Definition
| Like chromosomes, it will need a single origin of replication (ORi) |
|
|
Term
| Plasmids typically contain _____A making them useful vectors in cloning experiments |
|
Definition
| A) Origin of Replication ORi |
|
|
Term
| What happens when you just insert the piece of DNA you want replicated? |
|
Definition
| nothing, it doesn't have ORi |
|
|
Term
| What can plasmids pick up? |
|
Definition
| A piece of foreign DNA without ORi |
|
|
Term
|
Definition
| To protect your DNA of interest from being digested by the cell |
|
|
Term
| What else can plasmids carry that can benefit the distinction of the growth of YGI? |
|
Definition
| most carry at least one antibiotic resistance gene. Adding an antibiotic will kill off any organism without that resistance |
|
|
Term
| Cloning vectors also have a number of _____ _____ ______ |
|
Definition
|
|
Term
| How did Cohen and Boyer determine that their cloning of DNA was successful? Refer to figure 4.2 |
|
Definition
| One plasmid had resistance to Tet, the other to Strep. Adding them together brought both resistances together causing a more resistant plasmid than original (tet and strep) |
|
|
Term
| What are the 4 steps to DNA cloning? |
|
Definition
1. Cut both vector and DNA fragment with same restriction enzyme(s)
2.2. Use DNA ligase to paste them together
3.3. Transform ligated DNA mix into bacterial host (usually E.coli)
4.Grow E.coli on antibiotics to screen clones/colonies with recombinant DNA |
|
|
Term
| What is a problem with cutting using the same restriction enzyme and what is a way around this problem? |
|
Definition
| you may have multiple ways in which the DNA can fit in plasmid in different directions (like upside down), a way around this problem is to use 2 different enzymes on 2 different sides of the plasmid, IE Bidirectional cloning |
|
|
Term
| What are 2 methods of transformation into the bacterial host? |
|
Definition
| Chemical transformation (with heat) or electroporation (shock) |
|
|
Term
| Is transformation successful with linear DNA? |
|
Definition
|
|
Term
| How many plasmids can each bacterial host take up? |
|
Definition
|
|
Term
|
Definition
| collection of clones that covers an entire source (like a chromosomes in fragments) collection of entire starting material except it is in fragments and cloned into plasmids |
|
|
Term
| what is a way to get around the disruption of a certain antibacterial resistance? IE determining what Plasmid has the YGI |
|
Definition
| Looking for absence of growth AKA replica plating |
|
|
Term
|
Definition
| Making an identical plate to determine what growth has ceased compared to one that has not when you disrupt an antibiotic quality of a certain plasmid |
|
|
Term
|
Definition
It codes for Beta-galactosidase
It breaks down a disaccharide (lactose) -->into Galactose + glucose
Used as a selection tool |
|
|
Term
| Complete the sentence: The growth media for post trans, contains 2 relevant compounds ___+____ |
|
Definition
|
|
Term
|
Definition
| A lactose analouge that can induce the lacZ gene, this INDUCES (INDUCER) the gene expression |
|
|
Term
|
Definition
A colorless compound that is cleaved by B-galactosidase into a blue dye
Xgal-->Galactose + Blue |
|
|
Term
| If the lacZ gene is cleaved by a plasmid what would the appearance of the bacterial colonies with the YGI in it look like? blue or white? |
|
Definition
|
|
Term
| If the bacteria contain plasmid w/o YGI the colonies will be... |
|
Definition
|
|
Term
| What are some reasons that the Blue/White assay can give false positives? |
|
Definition
Mutations: point mutation during replicatoin.
Deletion--at some point part of the gene is deleted, Exonuclease contamination (lacZ gene may get chewed up and put back together) |
|
|
Term
| How can you prevent a false positive? |
|
Definition
| Working cleanly, carefully and in a sterile environment |
|
|
Term
| What is a method to prevent religation of a vector? |
|
Definition
| Use alkaline phosphotase to remove phosphate groups under alkaline conditions |
|
|
Term
| what do you have to be careful with when using alkaline phosphotase? |
|
Definition
| Don’t treat both your insert and your plasmid with Alkaline Phosphatase, otherwise they will never connect. |
|
|
Term
| How many base pairs can a bacteria clone? |
|
Definition
|
|
Term
| What is another method to get more BP cloned? |
|
Definition
|
|
Term
| How would you determine if your cloning was effective using bacteriophages? |
|
Definition
| You can take foreign DNA and insert into a phage and begin infecting it into bacteria and you would look for absence of growth where the bacteria have been infected and killed |
|
|
Term
| What are some advantages to using bacteriophages? |
|
Definition
1.Infect cells more efficiently
2.clones are not colonies, but plaques on a lawn of bacteria
3.Replace some phage genes with foreign DNA
4. can accept larger pieces of DNA
5. Often used to make genomic libraries |
|
|
Term
| What does mRNA do? What does cDNA do? |
|
Definition
mRNA-what genes are on
cDNA-what genes are expressed |
|
|
Term
|
Definition
| complementary DNA copy of RNA |
|
|
Term
|
Definition
| set of clones representing most/all of the mRNA of a given cell type at a given time |
|
|
Term
| What is the 1st step in cDNA cloning? and what is needed to do this? |
|
Definition
| Make DNA copy of RNA with Reverse Transcriptase |
|
|
Term
|
Definition
| RNA-Dependent DNA polymerase (telomerase) |
|
|
Term
| What is needed to initiate synthesis? |
|
Definition
|
|
Term
| Specifically what kind of primer is needed? |
|
Definition
A 3 prime hydroxyl
Put down by primase and a primer is needed, built on preexisting nucleotides |
|
|
Term
| What does RT make use of? |
|
Definition
| Poly-A tail on eukaryotic mRNAs, the poly-A tail is on every Eukaryotic organism's 3' end |
|
|
Term
| What is the Poly-A tail's primer? |
|
Definition
|
|
Term
|
Definition
1.start off with a strand of mRNA and add reverse transcriptase, and OligoDT
2.Reverse transcriptase will make a 3-->5' end of DNA
3.RNaseH will chew up the RNA and it will be left with a few fragments and then dNA polymerase will take over, so you need a primer for DNA polymerase, these fragments will be automatically created if RNase is used in small quantities.
4.DNA polymerase will now work from 5'-->3' using the primers to create the DNA. Other exonucleases that work in both 3-->5' and 5'-->3' directions will fix the strand up.
5. you will be left with a little RNA on the left remaining because there is no 3'-->5' primer any more for the polymerase to work... |
|
|
Term
| A break in the helix is called a ____ |
|
Definition
|
|
Term
| How can you remove a nick? |
|
Definition
| use DNA polymerase in the 5-->3' direction |
|
|
Term
| How do you add sticky ends where there are none? |
|
Definition
| Use Terminal Transferase + dCTP |
|
|
Term
| Where does Terminal Transferase + dCTP add? |
|
Definition
|
|
Term
| What is Terminal Transferase + dCTP used for? |
|
Definition
| creating a library, making complementary overhangs on a vector as well as the DNA strand |
|
|
Term
|
Definition
| Simple tool that revolutionized genetics and biotech. |
|
|
Term
|
Definition
| generates BIllions of copies of a single molecule of DNA in a test tube w/in hours |
|
|
Term
| What do you need for PCR (5 things) |
|
Definition
TAQ-polymerase
1. Template DNA (small amounts are sufficient) (small amounts)
2. Primers (forwards and Reverse)
3. Heat-stable DNA polymerase from Thermus aquaticus[lives in bizzare environments, hot springs, geysers etc] (TaqPolymerase)
4. Nucleotides (A,C,G,T)
5. PCR machine (AKA Thermal Cycler) |
|
|
Term
| What are the different temps in PCR and what do each do? |
|
Definition
95*C=separate strands of DNA
50*C=to ANNEAL primers (avoid melting and promote binding, must find ideal Tm based on # of base pairs and composition of strand)
72*C=for polymerase to extend strands
95*C=to separate strands and repeat |
|
|
Term
|
Definition
| Reverse Transcriptase PCR to make DNA from RNA |
|
|
Term
|
Definition
1.Start with mRNA instead of DNA template
2.Use RT to convert mRNA to single stranded DNA
3.Convert ssDNA to dsDNA
4.Continue with regular PCR |
|
|
Term
| what is Real-Time PCR (qPCR)? |
|
Definition
| Quantifies amplification of DNA as it is occurring in real time. |
|
|
Term
| What does qPCR use to detect the amount of DNA in each cycle? |
|
Definition
| fluorescent probes attached to quenching probes |
|
|
Term
| Where is the flurescent tag and quenching tag? |
|
Definition
|
|
Term
|
Definition
| during amplification the separated strands anneal to a reporter probe and then the fluorescent tag is released emitting light as DNA polymerase eats away at that bond |
|
|
Term
| What 4 things do you need to make a recombinant protein? |
|
Definition
cDNA of eukaryotic gene
Expression vectors
Inducible promoters
Expression systems (prokayotic vs. Eukaryotic [higher cost and more effort, low yield ]) |
|
|
Term
| What is an expression vector? |
|
Definition
| specialized vectors for expression of foreign genes |
|
|
Term
| What kind of promoters do expression vectors have? Why? |
|
Definition
Strong promoters b/c Control transcription,
Strong promoter yields a lot of transcript |
|
|
Term
| What else do expression vectors have? Why? |
|
Definition
Inducible promoters
Inducible promoters--one that can be controlled, turn on or off, you control when it is expressed or not expressed |
|
|
Term
| What is an inducible promoter ? |
|
Definition
| Control and regulate the transcription/expression of cloned gene |
|
|
Term
| Some examples of inducible promoters and what they are induced by. |
|
Definition
Lac promoter induced by IPTG
PBad promoter induced by arabinose sugar |
|
|
Term
| what are some disadvantages for using the lac promoter ? what is this? |
|
Definition
| Leaky Expression...Inducible promoters--one that can be controlled, turn on or off, you control when it is expressed or not expressed |
|
|
Term
| What are some advantages for using a fusion protein? |
|
Definition
.Easy purification of recombinant Protein
Use the Tag as a handle on something and using that tag to move it.
Affinity tag is a way to isolate YPI based on the presence of the tag
2. Increase expression levels and/or solubility of recombinant protein
a. You don’t want your protein to precipitation out of solution |
|
|
Term
| What are some disadvantages to using to using fusion protein |
|
Definition
| Additional protein sequence may affect structure and/or function of YPI |
|
|
Term
| What are the 2 main advantages of fusion proteins? |
|
Definition
1. Easy purification of recombinant Protein
2.2. Increase expression levels and/or solubility of recombinant protein |
|
|
Term
| What are affinity tags (fusion proteins) great for? |
|
Definition
| Purification/isolation, detection and localization |
|
|
Term
| Some examples of affinity tags? |
|
Definition
His6 (oligohistidine) (most popular and smallest, 6 histatine residues)
Must use an antibody to find it
GST (Glutathinone S-transferase (may be as large as your protein)
MBP(maltose binding protein) (also very big)
c-Myc
GFP (Green Fluorescent Protein)
The tag will fluoresce green! |
|
|
Term
| What helps begin the transcription Ptrc in a fusion protein? |
|
Definition
|
|
Term
| What is enterokinase (yellow) ? |
|
Definition
| A specific protease that is a counterpart to restriction endonucleases, these recognize a specific sequence and CLEAVE IT! |
|
|
Term
| Give some examples of proteases |
|
Definition
| TEV Protease and Tobaccotich Virus |
|
|
Term
| What happens when you expose the protein to EK (yellow)? |
|
Definition
| It will cleave the Yellow area removing the tag.. |
|
|
Term
| How do you purify a fusion protein (simplified version)? |
|
Definition
Transform
Lyse
Extract
Mixture Column
Elution agent
Protease
Mixture Column
TLEMEPM |
|
|
Term
| In detail describe how one would purify a fusion protein. |
|
Definition
1.Transform into bacteria
2. Extract Protein by Lysing Cells
3. Elute YPI off the column by adding something to compete with interaction, specifically IMIDAZOLE
4. Add Protease to separate YPI and Tag
5. Reapply to Mixture column |
|
|
Term
| What is the shorthand version of creating a fusion protein? |
|
Definition
| Shorthand version, ADD protease in the first mixture column step and collect flow through and then you have YPI, but in practice it is difficult to do |
|
|
Term
| What is transformation in prokaryotes? |
|
Definition
| introduction of foreign DNA |
|
|
Term
| What is transformation in eukaryotes? |
|
Definition
| conversion from normal to cancerous cell |
|
|
Term
| What term is to be used to introduce foreign DNA in eukaryotes? |
|
Definition
|
|
