(Nov 12-16) - Recombinant DNA Technology - Biology

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Dec 12, 2012 (4 years and 8 months ago)

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Recombinant DNA Techniques


The field of molecular genetics has been radically altered with the development of
techniques used to create
recombinant DNA molecules

and to
clone

(make
multiple

copies of)
those molecules. Since the discovery of these tools in the 1970s, many techniques for
manipulating and analyzing those molecules have also been developed. Using recombinant
DNA technology to manipulate genes for genetic analysis and for devel
oping products or other
applications is called
genetic engineering
. The goal of this lab is to learn about some of those
techniques used and to apply your knowledge in answering questions based on those concepts.


RESTRICTION ENZYMES


A
restriction enzyme

(or restriction endonuclease) recognizes a specific base pair
recognition sequence in DNA called a restriction site and cleaves the phosphodiester backbone
of the DNA within that sequence. Restriction enzymes are used to produce a pool of DNA
fragments.

Most restriction enzymes are found naturally in bacteria. In bacteria, restriction
enzymes protect the organism against viruses by cutting up invading viral DNA. More than 400
different restriction enzymes have been isolated. They are named for the org
anisms from which
they are isolated. A three
-
letter system is used; the letters are italicized, followed by roman
numerals. For example,
Eco
RI is from
E. coli

strain RY13.


Many restriction sites have an axis of symmetry through the midpoint. The
sequen
ce is often a palindrome, reading the same from left to right (5’ to 3’) on the top strand as
it does from right to left (5’ to 3’) on the bottom strand. For e.g., the restriction site sequence for
the
Eco
RI restriction enzyme is 5’ GAATTC 3’ (Fig. 1).
The most commonly used restriction
enzymes recognize four base pairs or six base pairs.
















Fig. 1: Cleavage of DNA by the restriction enzyme
Eco
RI.

This restriction enzyme from
E. coli

yields “sticky
ends” at the specific 6 base pair palin
dromic sequence shown. This yields fragments with single
-
stranded,
complementary ends.


Restriction enzymes cut DNA in different ways. Some enzymes cut both strands of DNA
between the same two base pairs to produce DNA fragments with blunt ends. Other e
nzymes
make staggered cuts in the symmetrical nucleotide pair sequence to produce DNA fragments
with sticky or staggered ends (Table 1).

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Table 1: Selected restriction enzymes and their recognition sequences.


GEL ELECTROPHORESIS

It is often desirable to separate DNA fragments from one another. This is usually
accomplished using
gel electrophoresis
, which separates the fragments on the basis of their
length. A mixture of DNA fragments is loaded at one end of a slab of agarose or
polyacrylamide
gel, which contains a microscopic network of pores. A voltage is then applied across the gel
slab. Over several hours, the DNA fragments become spread out across the gel according to
size.

Near neutral pH, DNA molecules carry a large negat
ive charge and therefore move
toward the positive electrode during gel electrophoresis. Because the gel matrix restricts random
diffusion of the molecules, molecules of the same length migrate together as a band whose width
equals that of the well into wh
ich the original DNA mixture was placed at the start of the
electrophoretic run. Smaller molecules move through the matrix more readily than larger
molecules, so that molecules of different length migrate as distinct bands (Fig. 2).
















F
ig.
2: Gel electrophoresis separates DNA molecules of different lengths.

A gel is prepared by pouring a liquid
containing a melted gel matrix between two glass plates a few millimeters apart. As the gel solidifies, a matrix
forms consisting of long, tangled

chains of polymers. The separated bands can be visualized by radiolabeling or
fluorescent tagging of the DNA.

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DNA bands on agarose or polyacrylamide gels are invisible unless the DNA is labeled or
stained in some way. One sensitive method of staining DN
A is to expose it to a dye that
fluoresces under UV light when it is bound to DNA. Another method involves incorporating a
radioisotope or a chemical dye into the DNA molecules before electrophoresis.


DNA SEQUENCING

In the late 1970s, researchers develop
ed methods that allow the nucleotide sequence of
any purified DNA fragment to be determined simply and quickly. Several techniques for
sequencing DNA have been developed, but the most widely used is the dideoxy method, which
is based on DNA synthesis carr
ied out in the presence of chain
-
terminating 2’,3’
-
dideoxyribonucleoside triphosphates (ddNTPs). These molecules, in contrast to normal
deoxyribonucleotides (dNTPs), lack a 3’ hydroxyl group (Fig. 3). Although ddNTPs can be
incorporated into a growing DN
A chain by DNA polymerase, once incorporated they cannot
form a phosphodiester bond with the next incoming nucleotide triphosphates. Thus
incorporation of a ddNTP terminates chain synthesis, resulting in a truncated daughter strand.
















Fig. 3
: Structures of deoxyribonucleoside triphosphates (dNTP) and dideoxyribonucleoside triphosphates
(ddNTP).

Incorporation of a ddNTP residue into a growing DNA strand terminates elongation at that point.


In this technique, DNA polymerase is used to make p
artial copies of the DNA fragment
to be sequenced. These DNA replication reactions are performed under conditions that ensure
that the new DNA strands terminate when a given nucleotide is reached. This produces a
collection of different DNA copies that t
erminate at every position in the original DNA and
differ in length by a single nucleotide. These DNA copies can be separated on the basis of their
length by gel electrophoresis, and the nucleotide sequence of the original DNA can be
determined from the o
rder of these DNA fragments in the gel.

Sequencing begins by denaturing a double
-
stranded DNA fragment to generate template
strands for in vitro DNA synthesis. A synthetic oligodeoxynucleotide is used as the primer for
four separate polymerization
reactions, each with a low concentration of one of the four ddNTPs
in addition to higher concentrations of the normal dNTPs. In each reaction, the ddNTP is
randomly incorporated at the positions of the corresponding dNTP, causing termination of
polymeriza
tion at those positions in the sequence (Fig. 4).



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Fig. 4: The use of ddNTPs causes chain termination.
A template strand of the DNA to be sequenced is
hybridized to a synthetic primer. The primer is elongated in a reaction containing the f
our normal dNTPs plus a
small amount of one of the four ddNTPs. Eventually the reaction mixture will contain a mixture of prematurely
terminated daughter fragments ending at every occurrence of the added ddNTP. The shortest fragment corresponds
to the 5’

most nucleotide sequenced.


Inclusion of fluorescent tags of different colors on each of the ddNTPs allows each set of
truncated daughter fragments to be distinguished by their corresponding fluorescent label. For
example, all truncated fragments that en
d with a G would fluoresce one color (e.g., yellow), and
those ending with an A would fluoresce another color (e.g., red), regardless of their lengths (Fig.
5).





















Fig. 5: Individual ddNTPs are labeled with fluorescent tags.

Four separate reactions can be performed, each
with a different ddNTP. If the ddNTP that terminates each truncated fragment is identified with a different
fluorescent dye, then all four reactions can be performed in the same reaction.

(For this picture
, ddATP = red,
ddGTP = yellow, ddTTP = blue, and ddCTP = green).


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The mixtures of truncated daughter fragments from each of the four reactions are
subjected to electrophoresis on special polyacrylamide gels that can separate single
-
stranded
DNA molecules d
iffering in length by only 1 nucleotide. It is often the case that all four
synthesis reactions can be performed in the same tube, and the products can be separated in a
single lane on a gel. A detector positioned near the bott
om of the gel reads and rec
ords

the color
of the fluorescent label on each band as it moves past, and a computer stores the sequence
for
subsequent analysis. Fig. 6

shows a sample printout (an electropherogram) from an automated
sequencer from which the sequence of the original tem
plate DNA can be read. The first
fragment detected will be labeled with the 5’ most nucleotide sequenced. The sequence analysis
will be given representing the newly sequenced strand in a 5’ to 3’ direction. Sequencing can be
now completely automated; ro
botic devices can mix the reagents and then load, run, and read the
order of the bases from the gel.


5’












3’

















Fig. 6
: DNA sequencing is now completely automated.

In an automated sequencing machine, the four reaction
mixtures are subjected to gel electrophoresis and the order of appearance of each of the four different dyes at the end
of the gel is recorded. Shown is a sample printout from an automated sequencer
from which the sequence of the
original template can be read
. (For this picture, ddATP = red, ddGTP = black, ddTTP = blue, and ddCTP = green).



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Names:__________________
____________

______________________________

______________________________

__________
____________________


Recombinant DNA

Group Activity (10 pts)


Activity

1

(1 pt)
:



You have been given chains of pop
-
beads meant to simulate truncated daughter DNA strands
generated by dideoxy sequencing.


A



red beads

G


yellow beads

T



blue beads

C


green beads


Simulate the arrangement of these DNA fragments on a polyacrylamide gel. Note where the
positive and negative electrodes will be positioned in gel electrophoresis. From the arrangement
of DNA fragments, determine the 5’ to 3’ nucleotide seq
uence of the newly synthesized strand
and the 5’ to 3’ sequence of the template strand.




Synthesized Strand:




Template Strand:




A
ctivity

2
:


1.
Your TA will hand you

electropherograms from 2 sequencing reactions. The data represents
sequencing of both strands of a protein
-
coding gene. From the data, determine the 5’ to 3’
nucleotide sequence of the newly made DNA strand in both reactions

and write it below in
the sp
ace provided

(1 pt).


A.





B.




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The two sequences represent a coding and a template strand of a gene in
E. coli
. A prokaryotic
promoter consensus sequence (
-
10 sequence: 5’ TATAAT 3’) is included in this sequence.


2. Determine which sequencing stra
nd was the coding or template strand of the given gene

(1
pt)
.








3. Determine the amino acid sequence encoded by this gene (it’s a short sequence of amino
acids!). Write that sequence below and include an indication of the amino and carboxyl
termini.

You can find an mRNA codon chart on Page 167 of your Lab Manual

(2 pts)
.








Below is a list of selected restriction enzymes and their recognition sites:


Restriction Enzyme

Restriction Recognition Sequence

Eco RI

5’ GAATTC 3’

䡩湤䥉f

5’
AAGCTT 3’

䵳Mf

5’ CCGG 3’

palf

5’ GTCGAC 3’

p睡f

5’ ATTTAAAT 3’

p牬ㄹrf

5’ TTTAAA 3’



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牥獴物捴楯渠敮sy浥m睯畬搠y潵⁵獥

⠱⁰琩
?







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牥獴物捴楯渠敮sy浥m睯畬搠y潵⁵獥

⠱⁰琩
?



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6. If you wanted to cut this DNA fragment into three smaller pieces, what restriction enzyme
would you use

(1 pt)
?







7.
You have some data

regarding the nucleotide sequence of this region of the genome, but
DNA sequencing is only accurate up to 500 bases. You would like to continue sequencing
the DNA. Using the sequence data given, design 2 primers that would allow you to continue
sequenci
ng both upstream and downstream of this sequence. Primers should be 20
nucleotides in length.

Do not forget to label the polarity of the primer ends.

(2 pts)


Sequence A Primer:




Sequence B Primer: