BSC 1010 Chapter Notes - Chapter 17: Polymerase Chain Reaction, Molecular Cloning, Recombinant Dna

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8 Feb 2017
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Chapter 17: Biotechnology (only sections 17.1, 17.2, 17.5, & 17.6)
Recombinant DNA: fragments of DNA from two different species, such as a bacterium & a mammal, spliced together in the laboratory
into a single molecule
Restriction endonucleases (restriction enzymes): are enzymes that cut a DNA molecule at a particular place
are the key to inserting a
sequence of DNA from one
organism into a piece of DNA
from another
the enzyme “scans” a DNA
molecule, looking for a
particular sequence, usually of
four to six nucleotides
was discovered from basic
research into why bacterial
viruses can infect some cells
but not others (referred to as
host restriction)
o bacteria produce
enzymes that can cleave
invading viral DNA at
specific sequences
o host cells protect their
own DNA from cleavage
by modifying bases at the
cleavage sites
o restriction enzymes don’t
cleave the modified
bacterial DNA
restriction sites:
locations on a DNA
molecule containing
specific (4-8 base
pairs in length) sequences of nucleotides, which are recognized by restriction enzymes
Restriction maps: is a map of known restriction sites within a sequence of DNA
Requires the use of restriction enzymes
In molecular biology, they are used as a reference to engineer plasmids or other relatively short pieces of DNA,
& sometimes for longer genomic DNA
Ability to cut DNA at specific places is important because
Allows physical maps to be constructed based on the positioning of cleavage sites for restriction enzymes
Provide crucial data for identifying & working with DNA molecules
Restriction endonucleases cleavage allows for the creation of recombinant molecules
Is critical to research because any steps in process of cloning & manipulating DNA require the ability to combine molecules
from different sources
There are 3 types of restriction enzymes
Type I & Type 3
Cleave with less precision & are not often used in cloning & manipulating DNA
Type II
Only one that cleaves in precise locations
Enable creation of recombinant molecules
o Recognize a specific DNA sequence, ranging from 4 bases to 12, & cleave the DNA at a specific site within this sequence
Recognition sites for most are palindromes (reads the same from 5’ to 3’ on one strand as it does on the complementary
strand)
o Cutting the DNA at the same base on either strand can lead to staggered cuts that produce “sticky ends”
Allow DNA molecules from different sources to be easily joined together
Although less common, some can cut both strands in the same position, producing blunt ends
o Can be joined with other blunt-cut ends
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Restriction endonucleases cut DNA into different size fragments
Most common separation technique used is
gel electrophoresis: a laboratory methods used to separate mixtures of DNA, RNA, or proteins according to molecular size
o molecules to be separated are pushed by an electrical field through a gel that contains small pores gel
gel is made of either agarose or polyacrylamide & spread thinly on supporting material
is submerged in buffer solution containing ions that can carry current & is subject to an electrical field
o takes advantage of DNA’s negative charge
string negative charges from the phosphate groups in DNA backbone cause it to migrate toward the positive pole
gel acts like a sieve
the larger molecules move slowly through the gel
smaller molecules migrate farther
once specific restriction fragment is isolated, it can be spliced into a plasmid that has been cut with the same restriction enzyme
DNA ligase is used to join the fragments together
It catalyzes the formation of phosphodiester bond between adjacent phosphate & hydroxyl groups of DNA nucleotides
Action of ligase is to seal nicks in one or both strands
Plasmids in isolation cannot replicate
To be useful, many identical copies must be generated & stably maintained
Molecular cloning involves the isolation of a specific sequence of DNA, usually one that encodes a particular protein product
(sometimes called gene cloning)
Transgenic organism: an organism into which a gene has been introduced without conventional breeding
Short sequences of DNA can be synthesized in vitro (in a test tube), the cloning of large unknown sequences requires propagation of
recombinant DNA molecules in vivo (in a cell)
Ability to propagate DNA in a host cell requires:
Vector: in molecular biology, a plasmid, phage, or artificial chromosome that allows propagation of recombinant DNA in a
host cell into which it is introduced
o Genomic library: representation of the entire genome in a vector
Genome is randomly fragmented
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