EXAM 3 QUESTIONS Flashcards
List the 3 basic components required for a bacterial cloning vector and describe the purpose of each.
- Origin of Replication:
ensures that the vector is replicated within the cells - Selectable markers: enable all cells containing the vector to be identified from those that don’t contain the vector
- Restriction sites: The point where DNA can be inserted into the vector. Needed so a DNA fragment can be inserted and replicated.
***** Look into slides
Describe, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.
1) The foreign DNA is cut by a restriction enzyme.
2) This fragment of DNA is then placed into the circular, bacterial DNA host (vector).
3) This plasmid that contains the foreign DNA is then ligated.
4) The plasmid is then able to replicate inside the bacterial host.
FROM THE SLIDES:
- Plasmid vectors are isolated and cut with a restriction enzyme.
- The DNA to be cloned is cut with the same restriction enzyme, producing a collection of fragments.
- These DNA fragments are spliced into the vector and transferred to a bacterial host for replication.
- Bacterial cells carrying plasmids with DNA inserts are identified by growth on selective medium and isolated.
- The cloned DNA is then recovered from the bacterial host for further analysis.
Answer the following questions about standard PCR:
- What enzyme is required for PCR? State the specific name of the enzyme, not just the type of enzyme.
- Apart from the enzyme, what 3 DNA molecules are required (give the technical names by which they are called)? What must be true about the sequences of these molecules? Note that dNTPs are individual nucleotides, not DNA molecules.
- Name the 3 basic steps of PCR and describe the molecular processes that occur in each. How is each step induced?
- How does this system work to amplify DNA?
- Taq Polymerase
- Single DNA strand, and 2 primers that must be complementary to the single strands.
- Denaturization.
High temperatures make the DNA separate into single strands.
Primers Anneal.
Low temperatures allow complementary primers to anneal to the single DNA strands.
Elongation.
Taq polymerase picks up at the primers and finishes the process of creating a complementary strand to the DNA.
This step occurs at an intermediate temperature, comparative to the other temperatures.
- Once the Taq Polymerase is finished, there is double the amount of DNA than there was at the start of the process. Thus the amount of DNA produced round after round increases exponentially.
One of the dominant features of the immune system is the capacity to generate new cells that contain different combinations of antibodies. Because there are billions of combinations it is impossible for each combination to be encoded by a single gene.
Explain in detail how such diversity is accomplished in the case of the light chain of a typical antibody.
Each mature B cell makes ONE type of light chain (kappa or lambda) and ONE type of heavy chain
An antigen stimulates a particular B cell with an antibody for that antigen
Produces population of plasma cells with antibody for that particular antigen.
The light chain of a typical antibody accomplishes diversity by being able to change based on any new threat.
expression vector
protein
What term is used to refer to the process in which DNA can be introduced into host cells.
Transformation
Of what advantage is it to have a polylinker region (Multiple unique restriction sites) embedded in the lacZ component in the pUC series of plasmids?
The lacZ gene is a selectable marker.
The colonies containing the newly inserted DNA will show up white because the LacZ gene will have been disrupted by the plasmid.
The normal colonies that don’t contain the new DNA will be blue in the presence of the Xgal in the agarose plate.
BamHI cuts the sequence 5’ G|GATCC 3’.
Which of the following sequences would NOT be recognized by this enzyme?
3’ TCCTTAAG 5’
(choose the option containing no
G next to another G)
Which of the following statements about ddNTPs is true?
They have a hydrogen at the 3’ carbon of the ribose sugar.
2nd and 3rd generation, also called next generation sequencing approaches…
(select all that apply)
allow high throughput parallel sequencing
allow to sequence the entire transcript content of cells
use a wide range of different technologies
Nucleic acid blotting is widely used in recombinant DNA technology.
In a Southern blot one generally….
Hybridizes filter bound DNA with a DNA probe
A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is
bacteria cannot remove Eukaryotic Introns
A human gene with a disease phenotype is going to be mapped by positional cloning.
Which would be most useful for this task?
Data about the inheritance of SNP markers in families with the disease.
Which of the following statements about manual Sanger sequencing is true?
The DNA sequence obtained is complementary to the template strand.
Assume that a plasmid (circular) is 3200 base pairs in length and has restriction sites at the following locations: 400, 700, 1400, 2600.
Give the expected sizes of the restriction fragments following complete digestion.
300, 700, 1000, 1200
When two proteins show a 50-70% match in amino acid sequence, it is likely…
The two proteins share a common ancestry
The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95°C.
Why would such a heat-stable polymerase be beneficial in PCR?
Each cycle includes a “hot” Denaturation phase (95°C), which Separates the Hydrogen Bonds that hold the strands of the template DNA together.
The human genome contains approximately 20,000 protein-coding genes, yet has the capacity to produce several hundred thousand gene products.
What can account for the vast difference in gene number and product number?
It is estimated that 40 to 60 percent of human genes produce more than one protein by alternative splicing.
Assume that one conducted a typical cloning experiment using pUC18, transformed an appropriate host bacterial strain (one carrying the lacZ complementing region), and plated the bacteria on an appropriate X-gal medium. Blue and white colonies appeared. Which of the two types of colonies, blue or white, would most likely contain the recombinant pUC18?
Explain your answer.
The white would most likely contain the recombinant PU18.
This is the study of “all genes in an organism in their entirety.”
genomics
What is a concise definition of proteomics?
the process of defining the complete set of proteins encoded by a genome
In a microarray experiment, a researcher has noticed that the mRNA expression of the KC gene is elevated in cancer cells of the prostate, compared to control prostate cells.
The researcher initiates the analysis of the KC gene.
What is the technical term for this strategy of a genetic approach?
reverse genetics
A mutation has been recovered in a mutational screen that appears to strongly enhance the progression of prostate cancer in mice.
A researcher wants to determine the role of the gene in normal and pathological conditions.
What is the term for this general strategic approach?
Forward Genetics
(starts with phenotype and goes to gene) asked TA
In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling the second step?
The purpose of the initial high temperature is to denature the DNA strand; the cooling step is required for the primer to anneal–this lower temperature is optimal for a primer to anneal (and create a free 3’ hydroxy end in order to bind DNA polymerase.)
What appears to be the range of number of protein-coding genes per genome in eukaryotes?
5,000-45,000
In what way are specific DNA sequences of the template amplified in the polymerase chain reaction?
In other words, how does one target the target?
The sequences at the ends of the target DNA sequence must be known.
Then primers that are complementary to these end sequences are added to the PCR system and allowed to anneal to the DNA at these specific sites.
This must happen in order for Taq polymerase to begin DNA synthesis.
Thus DNA synthesis can be targeted to begin at the primers that you add into the system.
Restriction endonucleases are especially useful if they generate “sticky” ends.
What makes an end sticky?
Single-stranded complementary tails
What is the specific application of reverse transcriptase in the preparation of cDNA?
to take a RNA sequence and create a complementary DNA sequence from it
What is the function of dideoxynucleotides in Sanger DNA sequencing? (multiple choice)
a. They act as primers for DNA polymerase.
b. They act as primers for reverse transcriptase.
c. They cut the sequenced DNA at specific sites.
d. They allow only the specific sequencing of the RNAs of a genome.
e. They stop synthesis at a specific site, so the base at that site can be determined.
They stop synthesis at a specific site, so the base at that site can be determined.
Which technique would NOT be used to find a gene for a functional protein in a sequenced region of a genome?
See if a SNP database contains sequences in the region.
There are different challenges that exist for sequencing prokaryotic and eukaryotic genomes. Which challenge is correctly paired with the type of genome to which it relates?
Eukaryotic: repetitive DNA
In the context of molecular genetics, reverse translation refers to
Assembling a DNA sequence from an amino acid sequence
A ddNTP, used often in DNA sequencing, lacks a(n) _________ at the __________ and _________ carbons.
OH, 2’, 3’
Restriction endonucleases…
cut DNA at specific sequences
List 2 especially useful characteristics of cloning vectors.
high copy number and antibiotic resistance gene(s)
What is the purpose of the LacZ gene plasmid cloning vector?
The LacZ gene is a selectable marker.
Acts as a reporter gene which encodes beta-galactosidase.
Expression of the lacz gene causes bacterial host cells carrying pUC18 to produce blue colonies when grown on medium containing a compound Xgal.
What is meant by the term low gene density?
Give an example of an organism with low gene density
Gene density is the number of genes per million base pairs.
Low gene density means that the percentage of a genome that encodes for something is relatively low. In other words, for a large genome, there are relatively few genes present. Instead, much of the genome might be non-coding, or might be repeat DNA. An example of such an organism with low gene density would be maize.
Electrophoresis separates DNA fragments of different sizes, but this technique does not indicate which of the fragments contains the DNA piece of interest. This problem is solved by ….
Removing the bands from the gel and hybridizing them with a known strand of DNA complementary to the gene of interest
The PCR (polymerase chain reaction) protocol that is currently used in laboratories was facilitated by the discovery of a bacterium called Thermus aquaticus in a hot spring inside Yellowstone National Park, in Wyoming. This organism contains a heat-stable form of DNA polymerase known as Taq polymerase, which continues to function even after it has been heated to 95°C. Why would such a heat-stable polymerase be beneficial in PCR?
Each cycle includes a “hot” denaturation phase (95°C), which separates the hydrogen bonds that hold the strands of the template DNA together.
mRNA
cDNA library
foreign DNA
transgene
lacZ
beta galactosidase
DNA quantification
real time PCR
taq polymerase
PCR
multiple hosts
shuttle vector
centromere
YAC
plasmid
cloning vector
protein
expression vector
chromosome spread
in situ hybridization
PCR is…
a technique for amplifying DNA sequences in vitro
The set of all proteins encoded by the genome is called the ________
proteome
Compared with prokaryotic chromosomes, eukaryotic chromosomes are
large, linear, less densely packed with protein-coding genes, mainly organized in single-gene units with introns.
Compared with eukaryotic chromosomes, bacterial chromosomes are
large, triple-heliz, Z-DNA, organized in monocistronic units with introns.
small, with high gene density
large, mainly unorganized in polycistronic transcription units without introns
large, mainly unorganized in monocistronic transcription units without introns
Small, with high gene density
Below are 4 processes common to most cloning experiments. Place components in the order in which they would most likely occur during a cloning experiment
Plating bacteria on selective medium
Cutting DNA with restriction endonuclease
Ligating DNA fragments
Transforming bacteria
- Cutting DNA with restriction endonuclease
- Ligating DNA fragments
- Transforming bacteria
- Plating bacteria on selective medium
Most of the bacterial genomes described in the text have fewer than
10,000 genes
One reason for generating a large number of clones in a eukaryotic genome library is that
each vector can take up only a relatively small fraction of the eukaryotic DNA
Name and describe 3 different kinds of bacterial cloning vectors
Plasmid cloning vector: It generally can load fragments of interest about 5-10kb long and it is derived from bacterial plasmids.
It has polylinker regions, selection system, insert discrimination system, and an origin of replication.
- Phage cloning vector: it can handle insert about 10-15kb; it is derived from bacterial virus (phage). To insert DNA fragments, the central third portion of the phage DNA, resulting in a left arm and a right arm and a central region. The fragments of interest then are inserted between the two arms, ligated by DNA ligase.
- Cosmid cloning vector: it can carry up to 50kb inserts; it is made of part phage and part plasmid, which makes it to be a engineered vector
A Linear DNA fragment is cut with a restriction enzyme to yield 2 fragments.
There is/are _____site(s) for this enzyme in the fragment.
1
A Linear DNA fragment is cut with a restriction enzyme to yield 3 fragments.
There is/are _____site(s) for this enzyme in the fragment
2
A Circular DNA fragment is cut with a restriction enzyme to yield 3 fragments.
There is/are _____site(s) for this enzyme in the fragment
3
In the context of recombinant DNA technology, what is meant by the term vector?
A vector is a vehicle to carry recombinant DNA molecules into the host cells where independent replication can occur.
What is meant by the designation EcoRI?
One of the first endonuclease enzymes isolated from strains of E.coli.
EcoRI is an endonuclease from E coli.
it is one of the first restriction enzymes isolated from E. coli
What 2 factors contribute significantly to the wide ranges of genome size among eukaryotes?
Number of introns and repetitive sequences
Variation in the number of genes
The first commercial production of what human enzyme led to the explosion of the biotechnology industry
Insulin
The Human Genome Project, which got under way in 1990, is an international effort to
Construct a physical map of 3.3 billion base pairs in the human genome.
Plasmids are important in biotechnology because they are…
a vehicle for the insertion of foreign genes into bacteria
What is the purpose of a cDNA library?
to produce a library of expressed genes
Which of the below are not steps in the production of genome sequence maps:
Read the sequence of individual piece of the genome
Identify molecular markers on specific chromosomes
When sequences are obtained, assemble and organize the sequences in order.
Isolate whole chromosomes
All of these steps you would use
Isolate whole chromosomes
Some vectors such as pUC18 and others of the pUC series contain a large number of restriction enzyme sites clustered in one region. What term is given to this advantageous arrangement of restriction sites?
complementation
palindrome
Beta-galactosidase
multiple cloning site
consensus sequence
multiple cloning site
What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?
It is a simple marker so that you can identify which bacteria contain the vector.
What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?
Endonucleases in bacterium might be used to break up the DNA injected into them by bacteriophages so that they can ward off infection.
or
Recognize viral DNA and digest it to protect bacteria’s DNA.
Of what advantage is it to have a polylinker region (Multiple unique restriction sites) embedded in the lacZ component in the pUC series of plasmids?
It makes it easy to identify what genes contain the recombinant DNA when grown on an x-gal medium (blue colonies don’t contain recombinant DNA, and white colonies do)
The lacZ gene is a selectable marker.
The colonies containing the newly inserted DNA will show up white because the LacZ gene will have been disrupted by the plasmid.
The normal colonies that don’t contain the plasmid will be blue in the presence of the Xgal in the agarose plate.
The increased restriction sites in the polylinker region increases the opportunity for the target DNA fragment to be inserted into the plasmid, and if embedded in the lacZ gene, it increases the likelihood that the gene will be interrupted by the insertion of the DNA. If the lacZ gene is interrupted, B galactosidase is not produced.
A section of a genome is cut with 3 enzymes: A, B, C.
Cutting with A and B yields a 10-kb fragment.
Cutting with B and C yields a 2-kb fragment.
What is the expected result from a digest with A and C, if the C site lies in between the A and B sites?
Only an 8-kb fragment
A set of overlapping DNA fragments that form a contiguous stretch of DNA is called a
contig
