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MolGen Final - Mod 4

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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.
pUC18 is a polylinker, which is characterized by increased multiple resritiction sites. The increased amount of restriction sties would increase the liklihood of the target DNA sequence being inserted into the plasmid. If the DNA sequence is insterted successfully and interrupts the LacZ gene sequence also in the resriction site, the bacteria will be unable to produce B galactosidase, which cleaves X-gal. When x-gal in the medium is not cleaved, the colonies turn white. If DNA was not successfully inserted into host DNA and the LacZ gene can produce B galactosidase, X-gal will be cleaved and the colonies will be blue. Because the white colonies will be the hosts containing the recombinant DNA, and pUC18 increases the likelihood of DNA being inserted, the white colonies will most likely contain the recombinant pUC18.
Answer the following questions about standard PCR:
1) What enzyme is required for PCR? State the specific name of the enzyme, not just the type of enzyme.
Taq polymerase
2) 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.
The complement, single DNA strand and 2 short oligonucleotide primers are required to flank each side of the target sequence. The sequence of the primers and complement DNA strand must be complementary to one another, so the primers can bind to the DNA.
3) Name the three basic steps of PCR and describe the molecular processes that occur in each. How is each step induced?
The first step of denaturization is induced by a high temperature, which facilitates the denaturing of double stranded DNA into single stranded DNA. The second step of hybridization/annealing is induced by a lower temp, which allows the primers to bind or anneal to the complement DNA, on either side of the target sequence. The final, third step of elongation is induced by a medium temperature, slightly higher than step 2, which allows the thermodynamically stable Taq polymerase enzyme to elongate the primers by synthesising new DNA.
4) How does this system work to amplify DNA?
Each run of PCR results in double the amount of initial DNA, thus after several runs, the initial target DNA is significantly amplified.
One of the dominant features of the immune system is the capacity to generate new cells that contain different combination of antibodies. Because there are billions of combinations it is impossibe for each combination to be encoded by a single gene. Explain in sufficient detail how such diversity is accomplished in the case of the light chain of a typical antibody.
Antibodies are made of segments. There are many different segments, each varying from one another. In the maturation of lymphocytes, segments are combined to form an immunoglobulin gene. The different possible uses of each particular segment create many possible combinations of the segments. The diversity is a direct result of somatic recombination on a single chromosome, where a v segment is moved to a position next to a J segment. After somatic recombination, the B cell is transcribed into mRNA containing one V gene, several J genes, and an additional C gene. This pre mRNA is then processed into a final RNA containing one of each each. This final RNA is then translated into a functional light chain. Each of these stages offer events which accomplish diversity.
Name at least 2 methods that are used to produce mutations in a forward genetics approach.
UV light, EMS, nitrosguanadine, transposons
What is meant by the term low gene density? Give an example of an organism with low gene density.
Low gene density is common in eukaryotes in which there may be as few as one gene in 64 kb base pairs, as is the case with a segment of human chromosome 22
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 increased restriction sites in the polylinker region increase the opportunity for the target DNA fragment to be inserted into the plasmid, and if embedded in the lacZ gene, it increases the liklihood that the gene will be interrupted by the insertion of the DNA. If the lacZ gene is interrupted, B galactosidase is not produced. When plated on X-gal medium, the resulting colonies containing the recombinant DNA will turn white from the inability to cleave X-gal with B galactosidase, as opposed to the blue colonies with a functional, uninterrupted lacZ gene. If the polylinker region is not embedded in the lacZ component, the liklihood that the recombinant DNA will disrupt the lacZ gene is not as high. The advantage creates an increased liklihood of a selective marker that determines the colonies that have recombinant DNA from those with nonrecombinant DNA, based on their color.
In the polymerase chain reaction, what is the purpose of the initial high temperature? What is the purpose of cooling in the second step?
In PCR, the initial high temperature serves to denature the double stranded DNA into single stranded DNA. Cooling in the second step allows for the annealing or hybridization of the primer to the DNA strand, flanking either side of the target DNA.
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would NOT be recognized by this enzyme?
[ ] a) 5′ AGGATCCGTA 3′
[x] b) 3′ TCCTTAAG 5′
[ ] c) 3′ CCTAGGATC 5′
[ ] d) 5′ AGCGGATCC 3′
Which of the following statements about ddNTPs is true?
They have a hydrogen at the 3′ carbon of the ribose sugar.
In a three-point mapping experiment, how many different genotypic classes are expected?
8
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.
The difference between a genetic screening experiment and a selection experiment is that a screening experiment involves ________, whereas a selection experiment creates conditions that ________ irrelevant organisms.
visual examination, eliminate
What is the function of dideoxynucleotides in Sanger DNA sequencing?
They stop synthesis at a specific site, so the base at that site can be determined.
Which of the following are the important proteins needed for cloning a eukaryotic gene into a bacterial plasmid?
[ ] A. DNA polymerase
[ ] B. restriction enzymes specific for the target genes
[ ] C. DNA ligase
[x] D. Both B and C
Plasmids are important in biotechnology because they are
a vehicle for the insertion of foreign genes into bacteria
What properties must a molecule have to serve as a vector?
It should be able to replicate itself independently, contain a number of unique restriction sites that would enable the insertion of DNA fragments cut with the same enzyme, carry a selectable marker, and be easy to retrieve.
Which of the following statements about manual Sanger sequencing is true?
The DNA sequence obtained is complementary to the template strand.
The Human Genome Project, which got under way in 1990, is an international effort to
construct a physical map of the 3.3 billion base pairs in the human genome.
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.
Most of the bacterial genomes described in the text have fewer than
10,000 genes
A ddNTP, used often in DNA sequencing, lacks a(n) ________ at the ________ and ________ carbons.
OH, 2', 3'
Below are four processes common to most cloning experiments. Place components in the order in which they would most likely occur during a cloning experiment.
__1__ cutting DNA with restriction endonucleases
__4__ plating bacteria on selective medium
__2__ ligating DNA fragments
__3__ transforming bacteria
What is a concise definition of proteomics?
the process of defining the complete set of proteins encoded by a genome
Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?
single-stranded complementary tails
One of the primary reasons for generating a large number of clones in a eukaryotic genomic library is that
each vector can take up only a relatively small fraction of the eukaryotic DNA.
What appears to be the range of number of protein-coding genes per genome in eukaryotes?
5,000 to about 45,000
What is the enzymatic function of restriction enzymes?
to cleave nucleic acids at specific sites
List two especially useful characteristics of cloning vectors.
high copy number and antibiotic resistance gene(s)
Clones can be of a cell, an organism, or a molecule. What characteristic do they all have in common?
All are derived from a single ancestor
When two proteins show a 50 to 70 percent match in amino acid sequence, it is likely that
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, 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 degrees C. Why would such a heat-stable polymerase be beneficial in PCR?
Each cycle includes a "hot" denaturation phase (95oC), which separates the hydrogen bonds that hold the strands of the template DNA together.
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally
hybridizes filter-bound DNA with a DNA probe
Describe, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.
1. RE are used to cut both the plamid vector and the target DNA.
2. Combination of the vector and DNA results in some of the strands recombining with the complementary tails of the DNA annealing with those of the vector.
3. DNA ligase covalently binds the new segment into the vector.
4. the vector is introduced into a bacterial host where replication occurs.
5. Finally the target DNA is replicated and is able to be isolated from the vector once again using RE to cut out the target sequence for analysis.
One major difference between prokaryotic and eukaryotic genes is that eukaryotic genes can contain internal sequences, called ________, that get removed in the mature message.
introns
What term is used to refer to the process in which DNA can be introduced into host bacterial cells?
transformation
What is a cDNA molecule?
A cDNA molecule is a DNA copy of an RNA molecule. Or complementary DNA
A principal problem with inserting an unmodified mammalian gene into a bacterial plasmid, and then getting that gene expressed in bacteria, is that
bacteria cannot remove eukaryotic introns
Which of the below are not steps in the production of genome sequence maps:
isolate whole chromosomes
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
A BLAST search is done to
find similar gene or protein sequences.
The first commercial production of what human enzyme led to the explosion of the biotechnology industry?
Insulin
Nucleic acid blotting is widely used in recombinant DNA technology. In a Southern blot one generally
hybridizes filter-bound DNA with a DNA probe.
It is common to use ddNTPs (dideoxyribonucleoside triphosphates) in which of the following biochemical reactions?
DNA sequencing
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?
multiple cloning site
This is the study of "all genes in an organism in their entirety."
genomics
What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?
(Isolated from bacteria, restriction endonucleases restrict or prevent viral infection by degrading the invading nucleic acid of the
What is the specific application of reverse transcriptase in the preparation of cDNA?
synthesis of DNA to form an RNA-DNA duplex
Fluorescent Sanger dideoxy sequencing methods uses what method to discriminate between the 4 different nucleotides?
Different fluorochromes attached to the four different ddNTPs.
Which of the following enzymes is used to make complementary DNA (cDNA) from RNA?
reverse transcriptase
Compared with prokaryotic chromosomes, eukaryotic chromosomes are
large, linear, less densely packed with protein-coding genes, mainly organized in monocistronic units with introns.
Match with the best letter choice:
expression vector

a. chromosome spread
b. protein
c. plasmid
d. centromere
e. multiple hosts
f. Taq polymerase
g. DNA quantification
h. protein/DNA interaction
i. lacZ
j. foreign DNA
k. mRNA
l. Agrobacterium tumefaciens
b. protein
What two factors contribute significantly to the wide ranges of genome size among eukaryotes?
The presence of introns and repetitive sequences is a major contributor, as are the differences in the number of genes.
Typically, bacterial DNA contains_____ (more or less?) repetitive DNA than eukaryotic DNA.
less
PCR is
a technique for amplifying DNA sequences in vitro
What is bioinformatics?
a method that uses very large national and international databases to access and work with sequence information
A human gene with a disease phenotype is going to be mapped by positional cloning. Which would be the most useful for this task?
Data about the inheritance of SNP markers in families with the diease
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
Compared with eukaryotic chromosomes, bacterial chromosomes are
small, with high gene density
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