Ch. 3 Q. 1,2,3,4,5,8,9,10
1. Type II restriction endonucleases, also known as restriction enzymes, are bacterial
enzymes that cut the DNA molecules internally at specific nucleotide locations to
produce discrete restriction fragments.
Recombinant DNA technology requires isolation of genes and transferring them from one
organism to another which is possible only if we are able to cut the source DNA
containing the target sequence and the cloning vector in appropriate positions to produce
discrete reproducible fragments which is achievable by using restriction enzymes.
2.
EcorI
BamII
HindIII
HaeII
EcorI
+
HaeII
EcorI
+
HindIII
EcorI
+
BamII
BamII
+
HindIII
BamII
+
HaeII
HindIII
+
HaeII
6.0
6.0
6.0
3.0
2.0
3.5
4.5
5.0
3.0
3.0
2.0
1.0
2.5
1.5
1.0
1.5
1.5
1.0
2.0
1.0
1.0
1.0
0.5
0.5
3. The two important considerations to be an efficient cloning vector are: the ability of
replication (must contain origin of replication) and presence of selectable markers such as
antibiotic resistance. Hence, pBR322 can be used as a cloning vector. The restriction
enzyme cuts the plasmid cloning vector pBR322 in a particular site to produce a linear,
sticky ended DNA which can be joined with the target DNA molecule which has the
same sticky end as the pBR322, after cleavage by the same restriction enzyme. Even after
cutting, the plasmid tries to reseal itself which can be avoided by adding alkaline
phosphatase, found in bacteria or extracted from calf intestine, and substitutes the 5’
phosphates with 5’ OH to prevent the unwanted ligation product or intramolecular
recircularization. However, the DNA molecule used for cloning has 5’ phosphate end
which can be ligated with the vector having 3’ OH in one of its strands by using T4 DNA
ligase. Due to the presence of 5’ OH (5’ phosphates converted to 5’OH by alkaline
phosphatase in one strand) two nicks are created after the joining. After transformation of
cloning vector-insert DNA into host cell, the nicks get repaired by the host cell DNA
ligase system.
A characteristic feature of pBR322 gene is the presence of two antibiotic resistance
genes- ampicillin and tetracyclin. It is 4361 base pair in length , and has unique BamHI,
HindIII, and Sa1I recognition sites within tetracycline gene. It also has a unique PstI site
in the ampicillin gene, a unique EcoRI site not included in coding DNA, and an origin of
replication that is functioning only in E.coli, with high copy number in E. Coli, and not
easy to transfer in other bacteria.
4. The pUC cloning vector is 2686 base pair in length having reverse oriented multiple
cloning sites (MCS). As in any other cloning vector, pUC vector also has: i) origin of
replication ( pMB1 replicon rep) ii) selectable marker for antibiotic resistance ( bla gene
which codes for B-lactamase which is ampicillin resistant) iii) Cloning site (Multiple
cloning sites (MCS) in opposite orientations). One important feature of pUC vector is the
presence of lac-Z region which contains CAP protein site, promoter Plac, lac repressor
binding site and 5’ terminal of lac Z gene which encodes the N-terminal fragment of Bgalactosidase. IPTG can induce the synthesis of this particular fragment, and forms blue
colonies if the substrate X-Gal is present. If the target DNA is inserted within the MCS
region in the lac Z gene, it inactivates the N-terminal fragment of beta-galactosidase and
gives rise to white colonies.
5.
a) We require that the restriction sites be evenly spaced in a genome rather than
occurring randomly. The restriction enzyme may have a larger area in one portion,
and tightly clustered area on some other part. So, we require different digestion
conditions for generating almost the same length of DNA fragment which is easier
with four base pair enzymes. Hence, the usage of two restriction enzymes (Sau3AI
and BamHI) becomes necessary. The important thing to be noticed here is that both
the enzymes have sticky end compatibility which is absolutely essential for the
ligation.
The necessity for using two different enzymes come from the fact that restriction
endonuclease sites are not randomly located and may create large fragments which
cannot be cloned due to which it may be difficult to find a specific target DNA
sequence. By usage of different restriction endonucleases, the problem can be solved.
b) Partial digestion refers to the digestion of a DNA by a restriction enzyme which is
blocked before the fragmentation of all the target sequences, and the main objective is
to produce overlapping fragments.
Partial digestion is usually achieved by reducing the incubation period, so the
enzyme does not have time to cut all the restriction sites, or by incubating at a low
temperature (e.g., 4°C rather than 37°C), which limits the activity of the enzyme.
c) In case of genomic libraries, partial digestion is essential to produce all the
intermediates of the fragments in order to produce overlapping fragments.
8. The different approaches used to detect a cloned target gene after creation of library
are: i) DNA hybridization, ii) immunological screening for protein product, iii) Protein
activity assaying, and iv) functional/genetic complementation.
DNA hybridization requires the formation of stable base pairs between the probe and the
target sequence. The virus having the recombinant DNA can be used to infect bacterial
cells. If we grow E.Coli on a regular petridish, the culture is turbid and cloudy,but E.Coli
incorporated with virus have clear regions in the petridish. A nitrocellulose filter paper,
which has the ability to bind with single stranded DNA, is placed on the petri dish for
around 30 secs to pick up the clear plaques. The filter is placed in alkali to get single
stranded phage DNA which gets attached to the nitrocellulose. Also, the single strands of
DNA probe (let’s assume cow DNA) are labeled with radioisotope or another tagging
system. If we mix the filter paper having the target DNA, and the cow DNA, then
hybridization/ base pairing occurs between complementary base pair sequences of the
DNA probe and target sample which can be detected by autoradiography.
Screening by Immunological assay requires the presence of protein, or part of it to be
determined by an immunological assay. Several master plates are used to grow clones of
the library, and the samples transferred to a known position in matrix to allow the
released protein to attach to the matrix. After the addition of primary and secondary
antibody, the uninteracting genes in the matrix are washed out. The addition of colorless
substrate, gets hydrolysed if secondary antibody is bound to primary antibody due to the
enzyme alkaline phosphatase and produces a colored compound. The colonies with
positive results are characterized to identify the gene of interest.
Screening by protein activity is used to identify the gene which produces an enzyme
which is not made by the host cell. The cells of the genomic library are plated on to a
medium containing a specific substrate, which if hydrolyzed gives rise to a colorimetric
reaction, and identifies the colony with the target gene.
Functional/genetic complementation is used when the gene of interest carries a
mutation that terminates the activity of the host cell enzyme. After the formation of DNA
library, the plasmids are transformed in the host cell with the deficiency. Now, only the
transformed cell with normal enzyme function is restored.
9. cDNA (complimentary DNA) Library refers to a nearly complete set of all the mRNAs
contained within a cell or organism. Working with mRNA is difficult since it is easily
degraded by RNAses, an enzyme called reverse transcriptase is used to produce a DNA
copy of the mRNA strand. These are called as cDNA ( reverse transcribed mRNA) which
collectively form the library.
In eukaryotic cells, the mature mRNA is spliced, and the cDNA lacks introns, therefore
allowing it to be expressed in bacterial cell. The bacterial cells can then be cultures to
make copies of the library for many experiments.
10. Plasmids are easier to handle than any other cloning vector. They are small, double
stranded, closed-circular DNA which contains origin of replication, which allows
replication independent of hosts genome, and selectable markers. It has straightforward
selection capabilities, and is used for cloning small DNA fragments (<10kbp).
The advantages of using cosmids and vectors derived from bacteriophages are that due to
the greater capacity of vectors large genes can be cloned, and also fewer clones of
genomic library have to be screened for a specific gene.
The bacteriophage  provides high efficiency in DNA packaging and infection process.
The specific advantage of the use of cosmids for cloning is that its efficiency is high
enough to produce a complete genomic library of 106 - 107 clones from a mere 1 pg of
insert DNA. Cosmids, which combines the properties of plasmid and bacteriophage 
vectors, can be used to clone very large DNA fragments (about 45kb), and maintained
like plasmids in E. Coli.
BAC (Bacterial Artificial Chromosomes) has the advantage of being the most
reproducible platform, and is used in genomic projects. The insert size is 150-350 kbp.