6.3 To further facilitate the separation heat is used (94 o C-96 ... - LCVI

twoeggfinnishBiotechnology

Dec 14, 2012 (4 years and 6 months ago)

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Molecular Genetics
-

Biotechnology

Chapter 6

Tools and Techniques


6.1



The United Nations Convention on Biological
Diversity defines biotechnology as:
"Biotechnology" means any technological
application that uses biological systems, living
organisms, or derivatives thereof, to make or
modify products or processes for specific use."


Specialized tools that need to be used by
molecular biologists to allow them to work with
DNA.


Recombinant DNA


晲慧浥湴f潦⁄ 䄠捯浰潳敤c
sequences originating from at least two different
sources

Tools and Techniques


Restriction Endonucleases



敮e祭y猠s桡琠慲h 汩k攠浯m散畬慲
獣s獳潲s 慮搠捵琠c乁 慴⁳灥捩a楣i扡獥⁰慩爠獥煵敮捥猠b牥r瑲t捴楯渠
敮e祭y猩⸠⁔ 攠捵e⁩猠愠 祤牯汹y楳⁲敡捴i潮o慴⁴桥h灨潳灨潤楥i瑥t
扯湤b


The site where the restriction enzyme works is called a
recognition site and it is usually palindromic and consists of four
to eight base pairs


Sticky ends


晲f杭g湴n敮搠潦⁡⁄e䄠浯m散畬e w楴栠獨潲s⁳楮杬攠
獴牡湤敤癥牨慮杳㬠
Eco
RI


More useful to molecular biologists as H bonds start to anneal naturally

Tools and Techniques


Blunt ends



晲慧浥湴⁥湤映愠䑎䄠
浯汥捵汥 睩瑨⁢慳敳⁦畬汹⁰慩牥搠⡦汵獨w
end);
Sma
I



Usefulness


䕣潒䤠桡猠愠獩砠扡獥⁰慩爠
牥捯杮楴r潮o獩瑥
潣捵牳s捥⁩渠敶c特r
4096 nucleotides = 4
6
)


not too many cuts to destroy whole
genes but enough cuts to make the
DNA workable



Tools and Techniques


Naming of Restriction Enzymes


BamHI


B


来湵猠
Bacillus


am


獰散楥i
amyloliquefaciens


H


獴牡楮


I


晩f獴 敮摯e畣汥慳攠楳潬i瑥搠晲潭⁴桥o獴牡楮

Tools and Techniques


Methylases & DNA Ligase


Methylases


敮e祭y猠瑨慴
慤搠愠浥瑨祬 杲潵瀠瑯湥t
瑨攠湵捬敯e楤敳i景畮搠楮⁡i
牥r瑲t捴楯渠敮摯湵捬敡獥
牥捯r湩n楯渠獩s攠敮獵物湧⁴桡琠
瑨攠灲潫慲祯a敳e潷渠䑎䄠摯敳d
湯琠来n 捬c慶敤


DNA Ligase


慮⁥湺祭攠
used to join together the
phosphodiester backbone of
DNA via a condensation
reaction


T4 DNA Ligase


敮e祭y 晲f洠
吴⁢慣a敲e潰桡来o瑨慴潩湳n
扬畮b

敮摳e瑯来瑨敲

Tools and Techniques


Gel electrophoresis



獥灡牡瑩潮映
捨慲来搠浯汥c畬敳渠瑨攠扡獩猠潦⁳楺攠
taking advantage of the chemical and
physical properties of DNA


DNA is negatively charged


敡捨e
nucleotide carries a phosphate group that
has a charge of
-
1


A charge is passed through the electrolyte buffer
solution with the negative end where the DNA is
loaded to the opposite positive end

Tools and Techniques


DNA can be cut with restriction enzymes


migration through the gel is inversely
proportional to the logarithm of their
size…that is, the shorter pieces move
further, the longer pieces do not move
very far


DNA is mixed with a loading dye
(visualization of DNA) and glycerol
(weighs DNA down so it will fall into the
wells).

Tools and Techniques


Gel is made from
agarose (seaweed
polysaccharide) or
polyacrylamide
(artificial polymer)


Gel gets stained with
ethidium bromide that
incorporates itself into
the rungs of the DNA
molecules and
fluoresces under UV
light.

Tools and Techniques


Bacteria often provide the appropriate
machinery (enzymes and ribosomes) for us
to produce proteins from a specific gene


楮獵汩渠數慭灬i


Bacteria have small circular pieces of DNA
called
plasmids

within their cytoplasm and
the relationship is endosymbiotic. Number
of base pairs ranges from 1000


200 000.


The number of plasmids in a bacteria is
known as the copy number; the more
plasmids, the more protein that can be created



Tools and Techniques


Transformation



楮瑲潤畣瑩潮 潦⁦潲敩杮e䑎䄬
批⁡b灬慳浩p爠癩牵猬⁩湴漠愠扡捴敲楡氠捥汬


Plasmids can be vectors that carry DNA to be
introduced into host cells

Tools and Techniques


Calcium Chloride Method

1.
Bacterial cells are suspended in 0
o
C, making the cell
membrane more rigid.

2.
Bacterial membrane contains exposed phosphates which are
naturally negatively charged.

3.
Positively charged calcium ions stabilize the negative
phosphates.

4.
Plasmid DNA is now introduced as the membrane is
chemically and physically “frozen”.

5.
The negatively charged phosphates of the DNA are stabilized
by the calcium
cations

6.
Shock treatment of 42
o
C for 90 seconds which causes the
outside of the cell to be warmer than the inside.

7.
This creates a draft which sweeps the plasmid into the
bacterial cell through its porous membrane.

8.
Environment goes back to 37
o
C.

Tools and Techniques


Electroporators



chambers that subject
bacteria to an electric shock which loosens
the structure of cell walls and allows DNA
to enter host cells.


Gene guns



water or He to shoot towards
DNA wrapped on a gold particle. The
force of the impact causes the DNA to be
propelled toward the target cells where it
penetrates cell walls and membranes.


Biolistic Particle Delivery System


Tools and Techniques


Selective plating

allows you to see if the
process has worked.


Attempt to grow new plasmid in an
antibiotic environment. If it grows, then
you know the plasmid was introduced as
the plasmid not only had the gene of
desire in it, but also an antibiotic
resistant gene in it.


Genetic Engineering


6.2


Genetic Engineering


慬瑥物湧r瑨攠獥煵敮捥映
䑎䄠浯汥捵汥D


First completed in the early 1970s by Cohen
(plasmids) and Boyer (restriction
endonucleases)


Insulin


㤰┠潦o摩慢整楣猠来琠瑨楳i灲潴p楮i
晲f洠扡捴b物慬r来湥瑩挠敮杩湥敲楮i


Somatropin


獩浩s慲 瑯⁧牯睴栠桯牭潮攠
獯浡s潴o潰楮i睨楣栠楳i畳敤⁴漠瑲t慴a
dwarfism and Turner’s syndrome


Also used to treat AIDS associated wasting
syndrome as it builds muscle

Advanced Techniques
-

6.3


Polymerase Chain Reaction


Mullis 1987
(p. 297, fig. 1)


PCR



數灯湥湴e慬⁡浰a楦楣慴楯渠潦⁄乁
sequence by repeated cycles of strand
separation and replication.


The methodology is closely related to DNA
replication:

1.
Two strands of DNA separated using
DNA
gyrase

and DNA
helicase
.

Advanced Techniques
-

6.3

2.
To further facilitate the separation heat is used
(94
o
C
-
96
o
C)


䠠H潮摳o来琠扲潫敮⁥慳楬礮


佮捥O獥灡s慴a 瑨攠楮i楶楤i慬a獴牡湤猠捡渠扥b
畳敤u慳a瑥浰污瑥t獴牡湤献


䑎䄠灲業敲猠慲D 畳敤⁩渠灬慣攠潦⁒乁⁰物o敲猠
⡄乁(牥灬p捡瑩潮t灲潣敳猩 慳⁄乁⁰物a敲猠捡渠
be easily created in laboratory settings.

5.
They are complementary to the opposing 3’
-
5’ends to the DNA target sequence to be
replicated.

6.
The temperature is dropped to 50
o
C
-
65
o
C for
the primers to anneal with the template DNA.

Advanced Techniques

7.
T
aq
polymerase (a high temperature DNA
polymerase) adds complementary nucleotides.

8.
Process takes place at 72
o
C which
Taq

polymerase can withstand as it is a DNA
polymerase that has been isolated from
Thermus aquaticus

(hot springs bacteria).

9.
The target area is not completely isolated in the
first few cycles of DNA replication and
Taq

polymerase adds nucleotides until it reaches the
end of the DNA.

Advanced Techniques

10.
Variable length strands are produced that start at the
target region at one end and extend beyond the target
at the other end.

a.
In the second cycle the DNA strands are again
heated and separated and primers allowed to
anneal. On two of the DNA strands one end
terminates at the target region (from cycle one) and
the primers anneal to the other end of the target
area.

b.
Taq polymerase

then adds appropriate nucleotides
and ceases when it reaches the end that terminates
with the target region. These are constant
-
length
strands.

c.
By the third cycle the number of copies of the
targeted strands increases exponentially

Advanced Techniques


Restriction Fragment Length Polymorphism


Polymorphism


any difference in DNA
sequence, coding or noncoding, that can be
detected between individuals



Restriction fragment length polymorphism
(RFLP) analysis



瑥t桮楱i攠批e睨楣栠䑎䄠
牥杩潮猠慲攠摩来獴d搠畳楮朠牥獴物捴楯渠敮穹浥猠
慮搠獵扪散瑥搠瑯t牡摩潡捴楶攠捯浰汥浥湴慲礠
DNA probes to compare the differences in
DNA fragment lengths between individuals.

Advanced Techniques


Southern blotting


灲潣敳猠瑨慴t慬汯睳⁄乁
牵渠潮⁡o来氠瑯t扥⁴牡湳晥牲敤e瑯t愠湹汯渠
浥浢牡湥n畳楮朠敬散瑲楣t氠捵牲敮琮


This is then placed in a solution containing
radioactive complementary nucleotide probes.


Base paring between the DNA and the probes
is called hybridization.


After hybridization the blot is exposed to x
-
ray
film and areas that have hybridized will band
out…this is called an autoradiogram.


Advanced Techniques


DNA Sequencing


Human Genome Project



卡湧敲
摩d敯硹

浥瑨潤m潲⁃ 慩渠呥T浩m慴楯渠呥T桮楱i攠
(1977)…DNA sequencing technique based on
DNA replication that uses
dideoxy

nucleoside
triphosphates


DNA template is treated as being single
stranded


A short single stranded radioactively labeled
primer is added to the end of the DNA
template

Advanced Techniques


Identical copies of this
complex are placed in four
reaction tubes


Each tube has DNA
polymerase and a supply of
all four deoxynucleoside
triphosphates (dATP; dTTP;
dGTP & dCTP)


Also, each of the four tubes
contains a different
radioactively labeled dideoxy
analogue (nucleoside
triphosphate whose ribose
sugar does not possess a
hydroxyl group on the 3’
carbon)

Advanced Techniques


What does this do?


Well, if the 3’
-
OH group is missing (as it is on the ddNTPs)
then the phosphodiester bonds can not be continued and the
chain will stop growing when one of the ddNTPs are
incorporated. Since only a fraction of the dNTPs are dideoxy
analogues, different lengths of DNA will be built. Take the
following example from the tube that contained ddATP:



5’


TTA




TTACGTA




TTACGTACGTA




TTACGTACGTAA


3’


These differing lengths of DNA can be separate using gel
electrophoresis and analyzed accordingly.

Applications
-

6.4


Medical Applications


Genetic Screening


process by which an individual’s
DNA is scanned for genetic mutations


Personal information for later in life





Fetal scan


What do you do with this information?



Gene Therapy


慬a敲慴楯i 潦⁡⁧敮整楣⁳敱略湣 楮⁡i
organism to prevent or treat a genetic disorder


Chronic Pain


灲潮潣楣数瑩癥v瑲t湳浩瑴敲e 楮摵捥⁰慩渠慮搠
慮瑩湯捩捥灴楶p 瑲t湳浩瑴敲e 摡浰敮⁰慩d


The idea is to introduce antinociceptive transmitters into cells that
might cause pain or to areas that produce these transmitters already,
but with more genes they will produce more of the transmitter to
dampen the pain.


Applications


Agricultural Applications


Nestor and Chilton (1981) created the first transgenic
plant


Ti plasmid (tumour inducing) carried by soil bacteria
Agrobacterium tumefaciens was used as the vector


Bacteria enter the plant through a wound and create a
bulbous growth (crown gall)


Only the T section of the plasmid is incorporated into the plant’s
chromosomal DNA however a foreign gene that has been incorporated
into the T section therein gets incorporated into the plant’s
chromosomal DNA


Only dicotyledon plants (beans, peas, potatoes), monocotyledons
(wheat, corn, rice) require a gene gun to incorporate new DNA


Factors now transgenically inserted into plants: hardiness,
increased yield, uniformity, insect and virus resistance,
herbicide tolerance, antiripening and antiaging

Applications


Forensics


DNA from the crimescene is compared to DNA from
potential suspects


DNA fingerprinting


灡瑴敲渠t映扡湤n渠愠来氬⁦牯洠
RFLP or PCR that is unique for each individual


RFLP


牥煵楲敳e瑨慴⁴桥⁄乁 獡s灬攠楳⁵湤敧牡摥n


PCR


牥煵楲敳e潮汹楮畴i 煵慮瑩瑩敳e潦⁄乁 瑨慴⁣慮⁢攠
摥杲慤敤


Both utilize the non
-
coding regions of DNA as they are the locations
that the most variability lies. The non
-
coding regions differ in the
quantity of variable number tandem repeats


It is illegal in Canada to refuse to provide a DNA sample to
police on arrest if requested