Restriction enzymes were discovered in the 1960s and cut DNA at specific recognition sequences. There are five types of restriction enzymes that differ in subunit composition and sequence recognition. Restriction digestion leaves sticky or blunt ends that can be joined by DNA ligase. Transformation involves the uptake and recombination of extracellular DNA into bacterial cells through natural competence, chemical treatments, or electroporation. Genomic libraries contain fragmented, cloned DNA that represent an organism's entire genome.

1. Submitted By:
Deepika Kritaniya
Ph.D(BIOTECHNOLOGY)

2.  Restriction enzymes were first discovered in
the 1960s.
 In 1970 Hamilton smith discovered restriction
endonuclease from hamophilus influenza
strain.
 They occur naturally in bacteria where they
protect the bacterium against foreign
invading DNA from other organisms (e.g.
Viruses or phages)

3.  Type I One enzyme with different subunits for recognition,
cleavage, and methylation. Recognizes and methylates a
single sequence but cleaves DNA up to 1000 bp away. (eg.
SmaI, BamHI)
 Type II Two different enzymes which both recognize the
same target sequence, which is symmetrical. The two
enzymes either cleave or modify the recognition sequence.
(eg. HindII)
 Type III One enzyme with two different subunits, one for
recognition and modification and one for cleavage.
Recognizes and methylates same sequence but cleaves 24–
26 bp away. (eg. HaeIII , HindIII)
 Type IV recognize modified, typically methylated DNA and
are exemplified by McrBC of E.coli. Requires at least 2
recognition sites within a distance of 40 – 2,000
 Type V utilize guide RNAs to target specific non-
palindromic sequences found in organism. They can cut
DNA of variable length. The fexibility and ease of use of
these enzymes make promising for future genetic
engineering applications.

5.  Some restriction endonucleases cuts on the
two DNA strands, leaving two to four
nucleotides of one strand unpaired at each
resulting end.
 These unpaired strands are referred to as
sticky ends because they can base-pair with
each other or with complementary sticky ends
of other DNA fragments.
 Other restriction endonuclease cleave both
strands of DNA leaving no unpaired bases on
the ends, often called blunt ends.

7.  In 1967 Gellert Lehman, Richardson discovred DNA
ligase.
 E. coli and phage T4 encode an enzyme, DNA
ligase, which seals single-stranded nicks between
adjacent nucleotides in a duplex DNA chain.
(Olivera et al.1968, Gumport & Lehman 1971).
 The T4 enzyme requires ATP, while the E. coli
enzyme requires NAD+. In each case the cofactor is
split and forms an enzyme–AMP complex. The
complex binds to the nick, which must expose a 5′
phosphate and 3′ OH group, and makes a covalent
bond in the phosphodiester chain.

9.  Natural transformation is a mode of
horizontal gene transfer (HGT) in bacteria
that contributes to the maintenance and
evolution of bacterial genomes.
 Since the discovery of natural transformation
of Streptococcus pneumoniae competent
cells, (Griffith, 1928), and the identification of
DNA as the transforming principle by Avery,
MacLeod and McCarty in 1944 (Avery et al.,
1944).

10.  More than 90 bacterial species have been
shown to be naturally competent eg; B.
subtilis, S. pneumoniae, and Haemophilus influenza,
Neisseria gonorrhoeae , H. pylori etc. (Lorenz and
Wackernagel, 1994, Claverys et al., 2000,
Dubnau, 1999, Johnsborg and Håvarstein,
2009, Sørensen et al., 2005, Stewart and
Carlson, 1986).

11.  Transformation entails recombination of genes
between similar or different lineages, representing a
form of bacterial sex that increases standing genetic
variation, and the recombination that occurs as part
of this process is called chromosomal transformation.
 If the incoming ssDNA shares no homology with
recipient, but has a self-replication potential , it can
be reconstituted into its dsDNA form using
recombination and replication function. The
recombination that occurs as part of this process is
called plasmid transformation or viral transfection
(Viret et al., 1991, Chen and Dubnau, 2004, Claverys
et al., 2000, Claverys et al., 2009, Dubnau, 1999,
Smith et al., 1995, Stewart and Carlson, 1986).

12.  Chemical transformation method utilizes
CaCl2 and heat shock at 42°C to promote
DNA entry into cell.
 CaCl2 causes the DNA to precipitate onto the
surface of the cell.
 Perhaps the salt is responsible for some kind
of change in the cell wall that improves DNA
binding.

13.  It use a short pulse of electric charge to
facilitate DNA uptake.
 Electroporation induces formation of
microscopic pores within a biological
membrane.
 These pores allow molecules ions and water
to pass from one side of the membrane to
other.

14. When DNA (the bacterial chromosome) is extracted for
transformation experiments, some breakage into
smaller pieces is inevitable. If two donor genes are
located close together on the chromosome, then there
is a greater chance that they will be carried on the
same piece of transforming DNA and hence will
cause a double transformation.

15.  A DNA library is a collection of DNA clones,
gathered together as a source of DNA for
sequencing, gene discovery or gene
function study.
 Among the largest type of DNA library is a
Genomic library.
 Genomic library which contains the entire
Genome (exons and introns).

16.  The first step in preparing a genomic library is
partial digestion of the DNA by restriction
endonuclease, such that any given sequence will
appear in fragment of a range sizes- a range that
is compatible with the cloning vector.
 Fragments that are too large or too small for
cloning are removed by centrifugation.
 The cloning vector such as BAC or YAC plasmid,
is cleaved with the same restriction endonuclease
and ligated to the genomic DNA fragments.
 The ligated DNA mixture is then used to
transform bacterial or yeast cell to produce a
library of cell types.

17.  Ideally all the DNA in the genome under study
will be represented in the library.
 Transformed bacterium or yeast cell grows into a
colony or clone, of identical cells bearing the
same recombinant plasmid.
 Cells containing particular DNA sequence can be
identified by DNA hybridization methods.
Hybridization method can order individual clones
in library by identifying clones with overlapping
sequences.
 A well characterized library may contain
thousands of contigs.

20.  Generally higher capacity vectors were used
for cloning such as cosmids, BACs, PACs, and
YACs.
 Advantages of such vectors is that the
average insert size is much large.
 The number of recombinants that need to be
screened to identify a particular gene of
interest is correspondingly low.
 Genes are more likely to be contained within
a single clone and fewer clones are required
to assemble a conting.

21.  http://webdoc.nyumc.org/nyumc/files/sun-
lab/attachments/CPMB.Ch.05.library.construct.pdf
 https://www.google.co.in/search?q=dna+blunt+en
ds&biw=1440&bih
 http://www.ncbi.nlm.nih.gov/books.
 Principles of Gene Manipulation - S. B.
Primrose, Richard M. Twyman, R. W. Old e-
book.
 https://archive.org/details/LehningersPrincip
lesOfBiochemistry5.

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