Describe, in order, the steps usually followed in producing recombinant DNA molecules in a plasmid vector.
-plasmid and the DNA molecule you're trying to clone are cut by the same restriction enzyme
-DNA fragment anneals to the sticky end of the plasmid with the help of DNA ligase
-the plasmid is introduced into a bacterial cell by one of several methods, including heat shock
-the plasmid is able to replicate within the cell and it replicates the foreign (recombinant) DNA along with its own genes
-the bacterial cells with the recombinant DNA are usually grown in a medium with antibiotic, along with normal cells
-the normal cells die, but the recombinant cells have antibiotic resistance, thanks to the genetic makeup of the plasmid
What is the specific application of reverse transcriptase in the preparation of cDNA?
synthesis of DNA to form RNA-DNA duplex
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?
insert of DNA in polylinker inactivates lacZ component and allows identification of recomb plasmids under proper genetic and environmental conditions
What might be a reasonable function of restriction endonucleases in a bacterium, distinct from their use by molecular biologists?
restrict or prevent viral infections by degrading invading viral nucleic acid
BamHI cuts the sequence 5′ G|GATCC 3′. Which of the following sequences would NOT be recognized by this enzyme?
3′ TCCTTAAG 5′
Which of the following statements about ddNTPs is true?
They have a hydrogen at the 3′ carbon of the ribose sugar
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.
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
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.
__3__ transforming bacteria
__1__ cutting DNA with restriction endonucleases
__2__ ligating DNA fragments
__4__ plating bacteria on selective medium
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
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 such combinations it is impossible that each combination is coded by a separate gene. Explain in as much detail as you can how such diversity is accomplished in the case of the light chain of a typical antibody.
-Ab genes composed of segments (many copies of each segment, each slightly diff)
-segments joined to make immunoglobulin gene
-many possible combinations b/c joining of segments is random
-diversity is result of somatic recombination, which moves V segment next to J segment
-B cell transcribed into pre-mRNA w/ 1 V gene, several J genes, 1 C gene
-pre-mRNA processed into mRNA, which is translated to functional light chain
-break and nibble mechanism also helps increase diversity
What are three key differences between a genomic and a cDNA library?
1) cDNA library enriched w/ fragments from actively transcribed genes
2) introns don't interrupt cloned sequences in cDNA library
3) cDNA libraries only contain sequences from mature RNA
Mario Capecchi, Sir Martin Evans, and Oliver Smithies recently won a Nobel Prize for gene targeting (gene knockouts) in mice. Describe the steps involved in creating a knockout mouse.
Step 1: A normal gene is disabled by inserting neo+ gene
Step 2: The disabled gene is transferred to embryonic mouse stem cells
Step 3: The disabled copy recombines with the normal gene on the mouse chromosome
Step 4: The cells are grown on a medium that contains the antibiotic G418. Only the cells with the neo+ gene will survive.
Step 5: Cells containing a neo+ gene are injected into early mouse embryos, which are implanted into a pseudopregnant mouse
Step 6: Variegated mice are then interbred and produce some progeny that are homozygous for the knockout gene
List the three basic components required for a bacterial cloning vector and briefly describe the purpose of each.
1) origin of replication - ensures vector is replicated w/in cell
2) selectable markers - enable cells w/ vector to be identified
3) unique restriction sites- where DNA fragments can be inserted
A pedigree and Southern blot results in humans are shown below. Filled-in figures represent individuals expressing a dominant trait (hypothetical) for blue tongue. What can you say about the potential linkage relationship between the allele responsible for the trait? Shaded regions within the homologs represent sequences that hybridize to the probe used for the Southern analysis. Arrows indicate cleavage sites used for the Southern analysis.
-mutant allele responsible for the dominant trait is very likely to be linked to homolog B.
-important because determining which chromosome is carrying a mutant allele for a trait is often an important first step in beginning the process of isolating the gene, mapping it on a chromosome, and so forth
-homolog B can also be used as a reliable marker for the disease and can thus be used for diagnostic testing.
List four uses of PCR
1) identification of RE variants
2) microsatellite analysis
3) screening for genetic disorders
Describe the basic components for a typical plasmid cloning vector system and the reason/use for those plasmid vector components.
-contains polylinker, ampicillin-resistance gene, origin of replication
-polylinker able to recognize several restriction enzymes, resistance gene allows for selective amplification, origin of replication so vector can replicate in host cell
Describe the standard PCR method (in sufficient detail) and how this process is able to produce clones in a "cell-free" system.
-target DNA is denatured using heat, giving two single strands
-Primers are added to flank the DNA of interest, and extended using Taq DNA polymerase.
-result is two new pieces of whole DNA containing the targeted gene.
-repeated 25-40 times to yield the desired amount of clones.
-all of these cellular machinery can run in this artificial environment, it does not require a host cell
The haploid human genome contains about 3 × 109 nucleotides. On average, how many DNA fragments would be produced if this DNA was digested with restriction enzyme PstI (a 6-base cutter)? RsaI (a 4-base cutter)? How often would an 8-base cutter cleave?
PstI would result in an average of 4.8 DNA fragments,
RsaI would result in an average of 76.9 DNA fragments.
An 8-base cutter would cleave every 8 65,536 bases, on average
Compare the fields of structural, functional, and comparative genomics. What is the purpose of each?
Structural= 3D structure of all proteins in a genome
Functional= interactions of DNA, proteins, and transcriptional factors
Comparative= functional genomics between species
Match each term with the best letter choice:
g.DNA quantification real-time PCR
a. in situ hybridization
b. expression vector
c. cloning vector
e. shuttle vector
g. real-time PCR
k. cDNA library
Rank from "roughest" to "fine detail" for the amount of resolution allowed by the following methods of mapping:
__4__ Cytogenetic map
__1__ Sequence map
__2__ Restriction map
__3__ Linkage map
Match the following terms with their definitions. Enter one number per box.
Synteny- Genes related by gene duplication in the genome.
Ortholog- Homologus genes of the same locus inherited from a common ancestor
Paralog- Conservation of the same groups of genes in the chromosomes of 2 or more species.
Homolog- Closely related genes based on sequence and function.
You have cut DNA from source A with restriction enzyme #1 and you have cut DNA from source B with restriction enzyme #2. Both of these restriction enzymes leave a 4 base single stranded overhang. You want to ligate these restricted fragments together. What must be true for this to be successful?
The single stranded overhangs must be complementary to one another and DNA ligase must be present to seal the fragments together.
Write the letter all of the following statements that are NOT true.
a. Coding sequences for gene products can be isolated from cDNA libraries.
b. Antibodies are used for Northern blot analysis. (screen mRNA)
c. VNTRs are highly conserved in human populations.
d. PCR amplification generates large numbers of linear DNA fragments.
e. RNA molecules can be used as hybridization probes in Southern blot analysis. (no, it's DNA)
b, c, e
A fragment of DNA is cloned into a plasmid with a sequencing primer binding site. After dideoxy sequencing, the gel pattern shown in this diagram is obtained. What was the sequence of the DNA strand that acted as the template in the sequencing reaction?
5' GCTAGCA 3'
Which technique would NOT be used to find a gene for a functional protein in a sequenced region of a genome?
see if SNP database contains sequences in the region
List two especially useful characteristics of cloning vectors.
1) high copy #
2) antibiotic resistance genes