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Exam 10: Gene Isolation and Manipulation

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Question 1

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A purified DNA preparation of a certain plasmid is digested to completion with BamH1 restriction endonuclease.In separate reactions,the same preparation was digested to completion with EcoRI and with a mixture of EcoRI and BamH1,respectively.The diagram below shows the appearance of the original molecules and the digestion products in the three digestion mixtures,after separation by electrophoresis in an agarose gel. Lane M-linear DNA markers of the indicated length in kb Lane 1-sample of original plasmid DNA preparation Lane 2-sample of the products of BamH1 digestion Lane 3-sample of the products of EcoRI digestion Lane 4-sample of the products of digestion of BamH1 + EcoRI Answer and justify the following: a)What was the original DNA molecule's length? b)Was the original plasmid DNA circular or linear? c)How many restriction sites for EcoRI and BamH1 did the original plasmid have? d)Draw the original plasmid,using B and E to indicate the location of the BamH1 and EcoRI restriction sites.Indicate the number of kb between sites.

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a)The original plasmid was 6 kb long sin...

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Question 2

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Use the following data to generate a restriction map for the circular "pEPP1" plasmid. PstI: 6.0 kb EcoRI: 6.0 kb SalI: 6.0 kb EcoRI + SalI: 2.0 kb,4.0 kb EcoRI + PstI: 1.5 kb,4.5 kb PstI + SalI: 2.5 kb,3.5 kb PstI + SalI + EcoRI: 1.5 kb,2.0 kb,2.5 kb

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Question 3

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The value of the beta-galactosidase gene in cloning could best be described as the gene:

Suppose you cut two different DNAs, $\begin{array} { l l } \text { one wih BamH1: } & \text { 5'-GGATCC-3' } \\& \text { 3'-CCTAGG-5' } \\\\\text { and one with BglII: } & \text { 5'-A GATCT-3' } \\& \text { 3'-TCTAG A-5' }\end{array}$ then ligate them together through their compatible sticky ends.Once joined,could you separate these two DNAs again with either restriction enzyme? Why or why not? (Each of these enzymes is cut between the first two nucleotides,at the 5? end of each strand of the palindrome.)

The restriction enzyme ClaI recognizes the sequence AT $\times$ CGAT.It cleaves the phosphodiester backbone between the T and the C bases at the arrow.The restriction enzyme TaqI recognizes the sequence T ↓ CGA.It cleaves the phosphodiester backbone between the T and the C bases at the arrow. a)Indicate the ends of the double-stranded DNA molecule left after cleavage by ClaI.Show the sequence,polarity,and overhang,if any. b)On average,how many sites would TaqI recognize in a random sequence of 1200 base pairs? c)If a TaqI-digested DNA end is ligated to a ClaI-digested DNA end,will ClaI cleave the newly ligated DNA? Will TaqI cleave the newly ligated DNA?

Which of the following statements about PCR is/are FALSE?

Bacterial artificial chromosomes are comprised of:

The polymerase chain reaction requires ssDNA primers that:

Transgenic plants can be generated using T-DNA plasmid carrying a gene of interest.To get the DNA into the plant cells,the researchers:

Thymidine kinase is valuable in the targeting of mouse genes because:

A certain animal virus contains a circular DNA that has five recognition sites for the restriction enzyme EcoRI.Infected cells make a large quantity of the viral surface protein.A cDNA corresponding to the mRNA for this surface protein has been cloned.How would you use a Southern blot to determine which EcoRI fragment or fragments contain the gene for the viral surface protein?

Perhaps the most common use of plasmid vectors is to:

A 3-kb supercoiled circular plasmid contains the restriction sites shown below.N and X represent NheI and XbaI sites,respectively;A through F mark six points on the plasmid.A preparation of plasmid DNA was separately digested to completion with XbaI alone,NheI alone,and a mixture of the two enzymes.The products were separated by agarose gel electrophoresis.Draw the predicted appearance of the gel,assuming that you ran the following in the various lanes. Lane M (size markers)-a mixture of known markers increasing in size from 1 kb to 6 kb,in steps of 1 kb Lane 1-sample from the original plasmid DNA preparation Lane 2-products of complete digestion with XbaI alone Lane 3-products of complete digestion with NheI alone Lane 4-products of complete digestion with XbaI plus NheI

A new restriction enzyme is discovered that recognizes an 8-base restriction sequence.About how many fragments of the Wombat genome (approximately 4.2 $\times$ 10⁸ in size)would you expect if you digested it with this enzyme?

This linear vector is comprised of bacteriophage genome components and can carry cloned DNA up to 15 kb in size.

Assume that a cosmid will carry inserts of about 50 kb and that cosmids are used to clone a 3 Mb (megabase) genome.Assuming you are particularly lucky and have no duplication in your library,what is the smallest number of cosmid clones you would need for a "complete" genomic library?

When seeking to identify the gene that is responsible for a particular cellular characteristic,scientists will generate cells carrying a recessive mutation that perturbs the characteristic of interest and then try to identify or "clone" the wild-type copy of that gene through functional complementation.This type of complementation could be described as:

You purified genomic DNA from tissue isolated from a population of roundworms (C.elegans).You wish to analyze this DNA,so you digest a portion of this DNA with the restriction enzyme EcoRI,and another portion of the DNA is digested with SalI.You make a gel made of agarose,with wells for loading your DNA.You place undigested DNA in well "A," the EcoRI digested DNA in well "B," and the SalI digested DNA in well "C." An electric field is generated by placing an anode (+ electrode)at the base of the gel,and a cathode (- electrode)at the top of the gel near the wells.The DNA migrates towards the anode. a)Describe the movement of the DNA along the gel.How is an electric field able to generate movement of the DNA fragments? b)How can nucleic acids be visualized in an agarose gel? c)How would you expect lane A,B,and C to appear? d)Can you analyze the size of fragments that contain a particular gene?

A DNA fragment produced by the restriction enzyme SalI is isolated from a digestion of mouse DNA.You cloned the fragment by first ligating it into a unique Sal I site found in a 3.5 kb cosmid.The ligation into the SalI site regenerates a SalI cut site at each end of the cloned fragment.You decide to determine the restriction map of this fragment.You digest the fragment with different restriction enzymes (below). Note: The cosmid used in this strategy does not have EcoRI or HindIII sites (but the cloned fragment apparently does). $\begin{array}{|l|l|}\hline\text { Restriction enzyme } & \text { Fragments produced } \\\hline \text { SalI } & 20 \mathrm{~kb}, 3.5 \mathrm{~kb} \\\hline \text { SalI }+ \text { EcoRI } & 7 \mathrm{~kb}, 13 \mathrm{~kb}, 3.5 \mathrm{~kb} \\\hline \text { SalI }+ \text { HindIII } & 4 \mathrm{~kb}, 5 \mathrm{~kb}, 11 \mathrm{~kb}, 3.5 \mathrm{~kb} \\\hline \text { SalI }+ \text { EcoRI }+ \text { HindIII } & 3 \mathrm{~kb}, 4 \mathrm{~kb}, 5 \mathrm{~kb}, 8 \mathrm{~kb}, 3.5 \mathrm{~kb}\\\hline\end{array}$

A wild-type Aspergillus strain is transformed with a plasmid carrying a hygromycin resistance allele,and cells are plated on hygromycin.One resistant colony showed an aberrant type of aerial hyphae.When crossed to wild type,the progeny were 1/2 normal hyphae,Hyg sensitive,and 1/2 aberrant hyphae,Hyg-resistant.The probable explanation is that: