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BIOL 239 Quiz: Set 17- Cloning
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8 Pages
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Department
Biology
Course Code
BIOL239
Professor
Christine Dupont

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Cloning
Summary:
o To reduce the size of large DNA strands, researchers use restriction enzymes
(endonucleases)
o These enzymes protect prokaryotic cells from viral infection by digesting viral
DNA. Bacteria shield their DNA from digestion by their own restriction enzymes
through the selective addition of methyl groups (CH3) to the restriction
recognition sites in their DNA.
o There are two ways in which enzymes make their cuts:
Blunt ends
Sticky ends
o Gel electrophoresis can be used to distinguish the different sizes of DNA
fragments
What is gel electrophoresis? Gel electrophoresis is the movement of
charged molecules (DNA) to an electrode
The electrodes cause the DNA molecules to migrate from one end to
another with size being a factor of the movement of molecules of DNA
Ethidium bromide is a fluorescent DNA binding added to the DNA which
helps the visualization of the DNA molecules
o Cloning fragments of DNA: Gel electrophoresis and restriction mapping can be
used for simple DNA molecules but the genomes of animals, plants and even
some microorganisms such as E. coli are too large and the digestion from one of
these organisms would result in too many fragments that could not be analyzed
In order to analyze these fragments, researchers must isolate and clone the
isolated molecules to analyze the isolated DNA fragment
Molecular cloning is a process that consists of two steps:
1. DNA fragments that fall within a specified range of sizes are
inserted into specialized chromosome-like carriers called vectors
which insure the transport, replication and purification of
individual inserts
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2. The combined vector- insert molecules are transported into
living cells and the cells replicate these molecules to form DNA
clones
o DNA clones can them be purified or stored within cells as
libraries
What are restriction enzymes?
o Endonucleases (enzymes that cleave phosphodiester bonds)
o that exist naturally in prokaryotes
o in the natural host, their function is to recognize foreign DNA sequences and cut
it into pieces for further degradation by host exonucleases
o restricts what type of DNA can exist in the host
o Like an immune system for prokaryotes (by luck)
o e.g., E. coli will accept the entry of DNA from other E. coli of the same strain, but
will destroy DNA from some phage
What do restriction enzymes recognize? How do they cut?
o Type II REN (Restriction Endonucleases) recognize inverted palindromic
sequences in DNA.
Each Type II REN has
a unique recognition
sequence.
o When they cut within
these sequences, they
leave either cohesive
(sticky) ends or blunt
ends
o palindromic sequences
are symmetrical and
when read 5' to 3' are
the same.
o Blunt ends cut right at
symmetry
Sticky ends cut at
beginnings and end of
palindrome, making
naked base pairs
Explain why EcoRI will cut
5'CGTT(GAATTC)GTACC3'
3'GCAACTTAAGCATGG5'
But not
5'CGTT(CTTAAG)GTACC3'
3'GCAA(GAATTC)CATGG5'
o EcoRI looks for the GA at the start and end, in the later version the CTT is at the
start so won’t work
o Remember read 5' to 3'
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Describe gel electrophoresis
o Gel electrophoresis distinguishes
DNA fragments according to size
Electrophoresis - the movement of charged
molecules in an electric field. DNA in
solution is weakly acidic (-ve charge) The
larger the DNA molecule, the more slowly
it moves through the gel matrix buffer for
current
o Mix with inert dye and glycerol for it sinks
Describe analyzing gel electrophoresis
o Circle blobs are tRNA - Similar size so not distinct, still binds to dye
o Bigger fragments don’t
move as much from well
since can’t get through
agarose
o Brighter fragments
should be bigger since
more regions for dye to
bind, but incomplete
digestion = faint
o Entire genomes =
millions of bands
(fragments), can’t be
well separated = smear
o Only small DNA
sections are used
Restriction Maps
o show the order of different size fragments
within small pieces of DNA
o consist of linear (or circular) diagrams of
the sites along a DNA segment at which
one or another restriction enzyme cleaves
the molecule
o Plasmid small circular DNA found in
many
o single-celled organisms
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Description
Cloning Summary: o To reduce the size of large DNA strands, researchers use restriction enzymes (endonucleases) o These enzymes protect prokaryotic cells from viral infection by digesting viral DNA. Bacteria shield their DNA from digestion by their own restriction enzymes through the selective addition of methyl groups (CH 3 to the restriction recognition sites in their DNA. o There are two ways in which enzymes make their cuts: Blunt ends Sticky ends o Gel electrophoresis can be used to distinguish the different sizes of DNA fragments What is gel electrophoresis? Gel electrophoresis is the movement of charged molecules (DNA) to an electrode The electrodes cause the DNA molecules to migrate from one end to another with size being a factor of the movement of molecules of DNA Ethidium bromide is a fluorescent DNA binding added to the DNA which helps the visualization of the DNA molecules o Cloning fragments of DNA: Gel electrophoresis and restriction mapping can be used for simple DNA molecules but the genomes of animals, plants and even some microorganisms such as E. coli are too large and the digestion from one of these organisms would result in too many fragments that could not be analyzed In order to analyze these fragments, researchers must isolate and clone the isolated molecules to analyze the isolated DNA fragment Molecular cloning is a process that consists of two steps: 1. DNA fragments that fall within a specified range of sizes are inserted into specialized chromosomelike carriers called vectors which insure the transport, replication and purification of individual inserts
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