56 terms

Module 4

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Compare the fields of structural, functional, and comparative genomics. What is the purpose of each?
Structural genomics- is concerned with organization and sequence of a genome

Functional genomics- characterizes what sequences or genes do

Comparative genomics- compares similarities and differences in gene content, function, and organization among genomes of different organisms
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
Describe the basic components for a typical plasmid cloning vector system and the reason/use for those plasmid vector components
Restriction sites: cut the plasmid and foreign DNA with the same enzyme and insert a gene.

Origin of replication: new recombinant genes will be able to replicate and increase in #.

Selectable markers: detect recombinant plasmids. Ex: lacZ gene produces B galactose but recombinant ones don't.
This term refers to the work undertaken by large teams of researchers who, through a concerted effort, clone and sequence the DNA of a particular organism
Genome project
A PCR technique that fills in small gaps by using the end of a cloned sequence as a primer to amplify into adjacent uncloned fragments
Primer walking
Rank from "roughest" to "fine detail" for the amount of resolution allowed by the following methods of mapping (1=roughest, 4=finest):

Cytogenetic map
Sequence map
Linkage map
Restriction map
Cytogenetic map- 2
Sequence map- 4
Linkage map- 1
Restriction map- 3
Match each term with the best letter choice: "cloning
vector"
plasmid
The smallest number of clones that represents the entirety of the genome are called what?
Minimum tiling path
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'
One of the primary reasons for the necessity of 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.
A typical prokaryotic genome has
1 million base pairs of DNA, containing 1000 genes.
Restriction endonucleases are especially useful if they generate "sticky" ends. What makes an end sticky?
single stranded complementary tails
The set of all proteins encoded by the genome is called the _______
proteome
In the genetic map of the human genome, one map unit is approximately 850,000 bp. For the genome of the eukaryotic yeast Saccharomyces cerevisiae, one map unit is approximately 3000 bp. What is a map unit, and why is it so different in these two different types of organisms?
A map unit is the amount of measured recombination b/w two link points. One map unit in a human is many more bp than in yeast due to the repitition in human genes. The amount of homologous pairs in humans must be lower than yeast.
As a model system, what are some of the advantages of the mouse as a model system?
Easy to grow, short generation time, readily matagenized and crossed

Mice have similar body plans and stages of development as humans

Similar genome size and number of chromosomes, as humans

Many genes have homologs in humans
Describe one (there's 3 options) major difference in the organization or content of prokaryotic and eukaryotic genomes
Eukaryotic genomes contain repetitive DNA that is largely absent in prokaryotic genomes

Genes are more densely spaced in prokaryotes verses eukaryotes

Prokaryotic genomes typically encode fewer genes than eukaryotic genomes
Match each term with the best letter choice: "PCR"
DNA quantification
What methods (can name 2) are used to produce mutations in a forward genetics approach?
UV light
EMS
Nitrosoguandandine
transposons
This is the study of "genes in their entirety."
genomics
During gel electrophoresis,__ will migrate more rapidly than __.
small DNA fragments, large DNA fragments
Match each term with the best letter choice: "ß-galactosidase "
lacZ
What are Northern analyses used for? Describe the steps involved in performing a Northern analysis, and describe how levels of gene expression are determined.
Northern blotting is a procedure used to transfer RNA from a gel to a solid support.
Step 1) RNA isolation
Step 2) Probe generation
Step 3) Denaturing agarose gel electrophoresis
Step 4) Transfer to solid support Step 5) Hybridization with probe Step 6) Washing Step
7) Detection
Levels of gene expression are determined by running a sample RNA on same gel to provide accurate sizing ladder or by the use of microarrays
The transcriptome of a genome contains more components than the proteome. Explain why this is true.
Transcriptome is all the RNA molecules transcribed from a genome which takes 3 codons. Proeteome is proteins encoded by the genome which is 3 codons to make one protein.
List four uses of PCR
Cell free cloning
paleobiology
forensics
Screening for genetic disorders
Why are telomeres and centromeres particularly difficult to sequence?
They consist of highly repetitive DNA, and so strand slippage issues can confuse the determination of a consensus sequence
The full-length (i.e., containing the entire protein coding region) cDNA for a specific eukaryotic gene in humans is 1500 nucleotides long. You screen a pig genomic library with this cDNA and isolate two genomic clones of different lengths. Both clones are sequenced and found to be 1900 and 2100 nucleotides long from start codon to stop codon. How would you explain the presence of two genomic clones in pigs, and the discrepancies in their length compared to the cDNA probe?
There was likely a duplication of this gene in pigs then the gene diverged from the pigs and humans or simply the humans lost a copy of the genes
Figure A below shows a restriction map of a rare prokaryotic gene with its direction of transcription indicated by the arrow. Figure B shows the unique cloning region (i.e., multiple cloning site) contained within a plasmid-cloning vector. The blackened region in Figure A represents the amino acid coding sequence of a protein that can be used in humans as a vaccine. The stippled region in Figure B is a highly active, constitutive (unregulated) prokaryotic promoter region. Letters indicate the cleavage sites for different restriction enzymes. Known gene sequences are indicated by short thick lines. Explain how you would isolate and fuse the coding region (Figure A) behind the indicated promoter in the cloning vector (Figure B) to produce large amounts of the protein in bacterial cells. Assume that the cloning vector carries the gene for tetracycline (an antibiotic) resistance. Letters represent different restriction enzymes
I would use restriction enzyme (RE) C with either RE D or RE B to clone the gene in the expression vector so that the beginning of this gene is directly 3' of the strong promoter.
You are handling a paternity lawsuit brought against five potential fathers by a woman. You isolated DNA from the mother, the child, and all the potential fathers. After using PCR to amplify specific polymorphic loci from each individual, you fractionate the amplified products on an agarose gel and stain with ethidium bromide to visualize the DNA fingerprints (shown below). Mo = mother; Ch = child; M1-M5 = potential fathers. Do these results confirm that any of the men are the child's biological father? Explain your answer
M4 could be the father. We can exclude all other males because the child has at least one band that is not present in the mother or any of the potential father except for M4.
Explain why the greatest diversity of human SNPs is found among African people
Humans are thought to have first evolved in Africa. These populations are the oldest with the greatest amount of time to accumulate polymorphisms
You determine that you have only three copies left of an important DNA fragment, so you decide to amplify it. Using flanking primers, how many PCR cycles would you have to run to generate over one billion (109) copies of the fragment?
28 to 30 cycles
Cloning reactions are done with DNA that has been cloned by restriction digestion and not by PCR. Using what you know about the way PCR works, why would you not want to use DNA from PCR to create DNA for cloning?
DNA polymerase makes a lot of errors when adding nucleotides. Also in PCR the Taq polymerase is not as accurate because it does not have a proofreading subunt. And if erros are made the ampiflication will be the wrong sequence.
What are three key differences between a genomic and a cDNA library?
Genomic contains both noncoded and coded DNA. Whereas cDNA only has transcribed regions. cDNA only contains sequences found in mature mRNA where the introns have been removed.
Describe the standard PCR method (in sufficient detail) and how this process is able to produce clones in a "cell-free" system.
PCR has two requirements: single-strand DNA template and a primer with a 3' -OH group.

First, begin with a solution that includes the target DNA, DNA polymerase, dNTP, primers, and magnesium ions in vitro.
Heat the solution to between 90 and 100 degrees Celsius, for about 1 minute, to break hydrogen bonds and produce two single-stranded templates. Quickly cool the solution to between 45 and 65 degrees Celsius, which allows primers to attach to template strands.
Heat solution to between 65 and 75 degrees Celsius, which Taq polymerase functions, replicating the region, and the entire process is able to be done in an in-vitro system, resulting in up to million of copies (clones) from a single DNA template target.
List at least three different kinds of bacterial cloning vectors
Plasmid, Phage, Bacterial artificial chromosomes (BAC), Expression Vectors
Explain why genetic and physical map distances may differ in relative distances between two genes on a chromosome.
Genetic map - less crossing over, more linkage.

The two loci are relatively close together on the same chromosome. The rate of recombination is proportional to physical distance, and since crossing over varies on different positions of the chromosome, the genetic map will just be an approximation of the location of the two linked marker loci.

Physical map - would express the actual distance between the two linked marker loci with greater accuracy.
Crossing over is often reduced around centromeric regions of chromosomes. If you were trying to construct a genetic map of two linked marker loci in this region, what result might you obtain and why? How would the genetic map correspond to the physical map?
Gene mapped based on recombination will appear to be very close together in centromeric regions due to low rates of recombination

Distances between the same genes on the physical map may be much greater when compared to their regions of the chromosomes
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.
The antibody genes are composed of segments. There are a number of copies of each type of segment, each differing slightly from the others.

In the maturation of a lymphocyte, the segments are joined together to create an immunoglobin gene.

The particular copy of each segment used is random, and because there are multiple copies of each type, there are many possible combinations of the segments

The diversity is a direct result of somatic recombination within a single chromosome, which moves one of the V segments to a position next to one of the J segments

In addition, the break and nibble mechanism plays a role in increasing diversity.
List the three basic components required for a bacterial cloning vector and briefly describe the purpose of each.
Origin of replication for propagation in the host

Selectable marker like Amp resistance

Polylinker or unique restriction enzyme sites to facilitate cloning
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 RE must have complementary sticky ends so that they will anneal together
Another word for a "DNA chip" (microscopic spots of oligonucleotides bound to glass that can be fluorescently labelled to identify levels of expression).
microarray
A map of the distribution of cloned genomic DNA from genomic clone libraries is called a...
Physical map
A map of the order, overlap and orientation of physically isolated pieces of the genome....
Physical map
A _______________ family is a group of evolutionarily related genes that arose through repeated evolution of an ancestral gene.
multigene
What is the purpose of an antibiotic resistance gene in a plasmid cloning vector?
To determine if the vector is present in host cell
A gene construct that d transcription occurs because the protein is easily identified (often GUS or GFP).
Reporter gene
What is a transgenic organism?
An organism that stably carries a foreign gene within is genome
The lungfish Protopterus aethiopicus has a genome 38 times larger than that of humans. Most of the DNA in this species is noncoding repetitive DNA. How could you create a library of clones that would let you compare just the genes in the lungfish to the genes in humans? 3-3
You could generate cDNA libraries and compare the transcribed regions of the genome
What is the purpose of the LacZ gene in a plasmid cloning vector?
To be able to distinguish those colonies with an insert using blue/white selection

white have an insert
Two genes that evolved from the same common ancestral gene, but are now found as homologs in different organisms are called _______________ .
orthologs
Match each term with the best letter choice: "in situ hybridization"
chromosome spread
Match each term with the best letter choice: "expression vector "
Protein
Match each term with the best letter choice: "transgene"
foreign DNA
Match each term with the best letter choice: "PCR"
Taq polymerase
Match each term with the best letter choice: "YAC"
centromere
Match each term with the best letter choice: "shuttle vector"
multiple hosts
Match each term with the best letter choice: "cDNA library"
mRNA