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WORK:1
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:- 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
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
Part B
Q4.How can DNA be radiolabled? Write different methods and elaborate at least one
of them.
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
KpnI
SmaI
NotI