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School: Department: Biotechnology


Name of the faculty member: Dr. Gyanesh Singh Course No: BTY655
Molecular Biology and
Course Title: Genetics
Class: M.Sc (Hon.), Biotech. Term:2nd Section: P7005 Batch:2010-12
Date of
Date of Allotment: Submission:
Max. Marks: 5 15/01/11 24/01/11

Submitted by:-
MANISH KUMAR SINGH
Roll no=08
Reg no=11006053
MSc (Hons) biotech
Q1.C, H, O, N, P are major components of DNA. How they interact with each other
to form double stranded DNA ?

Answer:- Carbohydrates are used as structural materials, transportable forms of


energy, or storage forms of energy. They consist of carbon, hydrogen, and
oxygen in a 1:2:1 ratio. Included in the carbohydrate are monosaccharides (has
two hydroxide groups bonded to the carbon backbone plus an aldehyde or
ketone group; carbon backbone), oligosaccharides (made of covalently bonded
monosaccharides), and polysaccharides (made of several covalently bonded
monosaccharides.

Lipids are nonpolar hydrocarbons used as energy stores, structural materials,


and signaling molecules. They consist of fats (1-3 fatty acids attached to a
glycerol unit), phospholipids (glycerol backbone, two fatty acid tails, a
hydrophilic head that contains a phosphate group and a polar group, and a
hydrophobic tail), sterols (lipids with no fatty acids, have a backbone of four
fused-together carbon rings), and waxes (long chain fatty acids taightly packed
and linked to long-chain alcohols or carbon rings).

Proteins function in nutrition, transportation, immunity, and signaling. They are


made up of amino acids (consists of amino group, carboxyl group, hydrogen
atom, and R group). Polypeptide bonds form between amino acids to form
polypeptide chains. Amino acid sequence is primary protein structure. The
secondary structure is the bonding pattern of the amino acids (e.g. helix, sheet,
etc.). The tertiary structure consists of the domain, where the sheets or helixes
fold on each other and become stable. The quaternary structure consists of
several polypeptide chains that form advanced proteins such as human
leukocyte antigens.

Nucleic acids consist of nucleotides (sugar, one phosphate group, N-containing


base). The different nucleic acids include DNA and RNA, and they are used in
storage and retrieval of heritable information
Q2.Why Type II RE are primarily used for restriction of DNA fragments?

Answer:- The central feature of type 2 restriction endonuclease is that each enzyme
has a specific recognition sequence at which it cuts a DNA molecule. A particular
enzyme cleaves DNA at the recognition sequence and now here else. For example ,
the RE called Pvu11 , cuts DNA only at the hexanucleotide CGATCG . Typical type
II restriction enzymes differ from type I restriction enzymes in several ways. They
are a dimer of only one type of subunit; their recognition sites are usually undivided
and palindromic and 4–8 nucleotides in length, they recognize and cleave DNA at
the same site, and they do not use ATP or AdoMet for their activity—they usually
require only Mg2+ as a cofactor. These are the most commonly available and used
restriction enzymes. In the 1990s and early 2000s, new enzymes from this family
were discovered that did not follow all the classical criteria of this enzyme class, and
new subfamily nomenclature was developed to divide this large family into
subcategories based on deviations from typical characteristics of type II enzymes.
These subgroups are defined using a letter suffix.

Type IIB restriction enzymes (e.g. BcgI and BplI) are multimers, containing more
than one subunit. They cleave DNA on both sides of their recognition to cut out the
recognition site. They require both AdoMet and Mg2+ cofactors. Type IIE restriction
endonucleases (e.g. NaeI) cleave DNA following interaction with two copies of their
recognition sequence. One recognition site acts as the target for cleavage, while the
other acts as an allosteric effector that speeds up or improves the efficiency of
enzyme cleavage. Similar to type IIE enzymes, type IIF restriction endonucleases
(e.g. NgoMIV) interact with two copies of their recognition sequence but cleave both
sequences at the same time. Type IIG restriction endonucleases (Eco57I) do have a
single subunit, like classical Type II restriction enzymes, but require the cofactor
AdoMet to be active. Type IIM restriction endonucleases, such as DpnI, are able to
recognize and cut methylated DNA. Type IIS restriction endonucleases (e.g. FokI)
cleave DNA at a defined distance from their non-palindromic asymmetric
recognition sites. These enzymes may function as dimers. Similarly, Type IIT
restriction enzymes (e.g., Bpu10I and BslI) are composed of two different subunits.
Some recognize palindromic sequences while others have asymmetric recognition
sites

Q3.What are major advantages of using Plasmids based vectors?

Answer:- A plasmid is a DNA molecule that is separate from, and can replicate
independently of, the chromosomal DNA.[1] They are double stranded and, in
many cases, circular. Plasmids usually occur naturally in bacteria, but are
sometimes found in eukaryotic organisms

Plasmids are considered transferable genetic elements, or "replicons", capable


of autonomous replication within a suitable host. Plasmids can be found in all
three major domains, Archea, Bacteria and Eukarya.[1] Similar to viruses,
plasmids are not considered a form of "life" as it is currently defined.[6] Unlike
viruses, plasmids are "naked" DNA and do not encode genes necessary to encase
the genetic material for transfer to a new host, though some classes of plasmids
encode the sex pilus necessary for their own transfer. Plasmid host-to-host
transfer requires direct, mechanical transfer by conjugation or changes in host
gene expression allowing the intentional uptake of the genetic element by
transformation.[1] Microbial transformation with plasmid DNA is neither
parasitic nor symbiotic in nature, since each implies the presence of an
independent species living in a commensal or detrimental state with the host
organism. Rather, plasmids provide a mechanism for horizontal gene transfer
within a population of microbes and typically provide a selective advantage
under a given environmental state. Plasmids may carry genes that provide
resistance to naturally occurring antibiotics in a competitive environmental
niche, or alternatively the proteins produced may act as toxins under similar
circumstances. Plasmids also can provide bacteria with an ability to fix
elemental nitrogen or to degrade recalcitrant organic compounds which provide
an advantage under conditions of nutrient deprivation

Part B
Q4.How can DNA be radiolabled? Write different methods and elaborate at least one
of them.

Answer:- A technique for conveniently radiolabeling DNA restriction


endonuclease fragments to high specific activity is described. DNA fragments are
purified from agarose gels directly by ethanol precipitation and are then
denatured and labeled with the large fragment of DNA polymerase I, using
random oligonucleotides as primers. Over 70% of the precursor triphosphate is
routinely incorporated into complementary DNA, and specific activities of over
109 dpm/μg of DNA can be obtained using relatively small amounts of precursor.
These “oligolabeled” DNA fragments serve as efficient probes in filter
hybridization experiments.

The present invention relates to a radiolabeled DNA oligonucleotide, a method of


preparation thereof and the therapeutic uses of this substance to prevent
uncontrolled cellular proliferation. The invention also relates to devices
incorporating the above radiolabeled DNA oligonucleotide for the therapeutic
treatment of uncontrolled cellular proliferation. More specifically, the present
invention is concerned with the prevention of restenosis by coronary delivery of
radiolabeled DNA oligonucleotide at a dilatation site of an artery. This invention
is also directed to a method of treatment of vascular proliferative diseases and/or
other proliferative disorders such as cancer and related metastasis. More
particularly, the invention relates to the preparation of DNA sequences carrying
one or several radioisotopes, located within the DNA sequence, and which are
able to prevent cell proliferation in vitro and, pursuant to local drug delivery,
are able to prevent cell proliferation in vivo, more particularly restenosis and
malignant tumors. In other words, the invention relates to the synthesis process,
the stability data of the radiolabeled DNA oligonucleotide, the efficacy of the
invention in vitro, in cell culture, and the in vivo delivery of the molecule.

Q5.What is the application of RNase H in biotechnology?

Answer:- RNase H is a non-specific endonuclease and catalyzes the cleavage of RNA


via a hydrolytic mechanism. Members of the RNase H family can be found in nearly
all organisms, from archaea and prokaryota and eukaryota.

RNase H’s ribonuclease activity cleaves the 3’-O-P bond of RNA in a DNA/RNA
duplex to produce 3’-hydroxyl and 5‘-phosphate terminated products. In DNA
replication, RNase H is responsible for removing the RNA primer, allowing
completion of the newly synthesized DNA

Function
In a molecular biology laboratory, as RNase H specifically degrades the RNA in
RNA:DNA hybrids and will not degrade DNA or unhybridized RNA, it is commonly
used to destroy the RNA template after first-strand complementary DNA (cDNA)
synthesis by reverse transcription, as well as procedures such as nuclease protection
assays. RNase H can also be used to degrade specific RNA strands when the cDNA
oligo is hybridized, such as the removal of the poly(A) tail from mRNA hybridized
to oligo(dT), or the destruction of a chosen non-coding RNA inside or outside the
living cell. To terminate the reaction, a chelator, such as EDTA, is often added to
sequester the required metal ions in the reaction mixture.
Q6.Write recognition sequence and ends produced by following RE
Sau 3AI, Eco RI, HpaII, KpnI, SmaI and NotI

Answer:- Sau 3AI:- recognition sequence

Eco RI:- recognition sequence


HpaII :- recognition sequence

KpnI

SmaI

NotI