CHAP 13 : TECHNIQUES IN BIOTECHNOLOGY

INTRO .

biotech uses cellular to make products tht are of to humans

.
processes use

traditional to bread alcohol

yeasts make &
•
: -

bacteria to make cheese &

yoghurt
modern treatment prevention of disease
•

: -

&

food prod
"

prod ' of clean

energy
of
enhanced efficiency manufacturing
-

processes
HUMAN GENOME

of DNA

1. POLYMERASE CHAIN REACTION amplify I multiply copies

target
"
seq .
of DNA
..
a

PCR technique used for of frm

in molecular bio
producing multiple copies DNA
sample
•

a .
a .

of of
segments DNA are
artificially multiplied through series repeated cycles of duplication using enzyme
•
a

in a thermo
cycler / PCR - machine
called DNA
polymerase

original
•

DNA template amplified to generate of of

can be millions copies original DNA
" '

INGREDIENTS

l . DNA template containing DNA region / gene of interest

to start off target sequence
short nucleotide sequence complimentary
2 .

primer ( oligonucleotide) brackets the

target seq .
tht need to be multiplied

segment of DNA complementary to targeted sequence of DNA

initiate the of new
↳
synthesisof
a

initiates replication by DNA

polymerase
strand DNA
°

complementary
 new DNA strands
( general )
>
synthesize
T rate of mutation when polymerase is working
( to vulnerable
polymerase enzyme )
3 * opens it be
. DNA up DNA & causes ,

naturally in
living organisms duplicate DNA when cells divide ( DNA replication )
°

occurs

binds to strand creates strand

single DNA &
complementary
•

type of
survive in hot env .
(s 70°C ) DNA polymerase
heat stable DNA extracted frm Thermos thermophilic bacteria Tag polymerase
polymerase aquatics
•
- =

4. nucleotides ( DNA nucleotides in

exceed DNTP
d ATP adenine

4 of bases
types
°

carrying
date guanine
d CTP
cytosine
↳ excess drips ( deoxy ribonucleotide triphosphate DTTP thymine

GUY
no cellular proof reading mechanism A heat
high ,
high rate
enzyme activity
↳
high rate of mutation of
higher pairing / mutations
↳ chance mis

j
-

✓
HEAT
bnrjdsrgdenwnbond ,

strands
ft eggett
°

exposes bases in each

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# 2

* Tag polymerase &

primers
stick I form hydrogen bonds are added

t
for primers to
Tag wnuokotides
attach

optimum temp .
aa.piiam.ea.ro ,

nucleotides to existing
bypariddimjnrg

by
allow Taq polymerase to attach free nucleotides
↳ cannot take by to attach if there's no starting primer qgq
one one
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APPLICATIONS

detect diseases
hereditary sickle-cell anaemia
phenylketonuria
•

, ,

fibrosis
cystic
°
detect viral diseases

forensic science
amplify DNA frm single drop of
•

blood ( semen ,
a strand of hair

amounts of extracted
tracing ancestry using small DNA
°

frm fossils

DNA profile / fingerprint

2. ELECTROPHORESIS & PROFILING -

banding patterns tht form

during
electrophoresis

•
a
person 's DNA can be used as identification
size of DNA fragments
profile / fingerprint
,
•
DNA DNA :
x
( unique ) sequence
-

technique tht uses the banding patterns of DNA fragments as identification

DNA
fingerprint is to particular indie
°

unique a .

method of
profiling ' '

( restriction enzymes ) at INGREDIENTS

using special enzymes
DNA cut
1 . is
specific
site of individuals
base
sequence recognition pieces of various
lengths I .
DNA

la
sticky restriction
-
ends
2 .

enzymes agarose gel modified w/

- wells

recognition site cut into one end of the gel

Hind TI

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.

( control to be compared w/)

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i t
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2 .
DNA
pieces were
separated by electrophoresis :

to
electrophoresis a process in which
charged molecules in solution are made
migrate
&
separate by passing an electric current through the solution

3 .
DNA pieces placed on a bed of semi -
solid
gel
4 of gel
. electric
passed through
current electrodes at either end
5 . DNA l
rely charged moves through gel toward
-
electrode + ve

6 .
smaller DNA
pieces move faster than larger DNA pieces ,

forms pattern of bands DNA

profile / fingerprint
= if wanna
compare baby &
of
mother
DNA ,
use same
type RE

Te a

wells

add DNA
"

longest fragments
of DNA

1500 bp

1000
bp slower
longer pieces
=

shorter pieces = faster

500 bp
✓

shorter fragments
+

+
+
of DNA
 I'
f :
← - :

& H
-
i

APPLICATIONS
•

tracing ancestry
DNA FINGERPRINTING
)
•

forensic science

identification of diseases
hereditary e.g cystic
•
.
,

fibrosis , Huntington 's disease

enables detection at
early provides greater chance tht condition be
effectively
-

an can
age ,

treated & possibly cured

determine the risk of affected child

having an
-

identification of alleles tht of disease

increase risk
developing
°

↳ colon cancer breast cancer

e.g
.

purpose
DNA SEQUENCING nucleotides target sequence / gene
} arrangement of DNA in a
determine the
precise
-

determination of the order of nucleotides of DNA

precise in
°

a
sample
'

nucleotides 1 bases

DNTP deoxy
ribonucleotide triphosphate
nitrogenous A
↳ DATP Adenine

a y n f y fs m i . g )
,
,
'

DGTP
¥:#
a ↳
nucleotides
ran .

free
' '

↳ DCTP ( unattached monomer )

↳ DTTP
5 prime end

Grime flee nuclettides DNA

'
end ' '

} in

'
-

catalysed by -

arranged as anti -

parallel
DNA polymerase r

strands

SANGER SEQUENCING

Sanger 's method of determining a DNA sequence

:

most frequently used

DNA is
synthesized frm 4 synthetic nucleotides ( a nucleotide
phosphate grps )
t 3 DNTP
•

however , Sanger sequencing reach also contain unique ingredient Di

deoxy nucleotides
•

a a or
,

chain -

terminating versions of all 4 nucleotides

REQUIREMENTS FOR DNA SEQUENCING

( DNA )
1 . DNA
template unknown

2 .

Tag polymerase
3 .
excess free nucleotides DNTPS

4. primers
+ EXCESS FREE DDNTP ( synthetic modified nucleotides )

Togop aq.RiboseE @AhM.di

°
deoxy nucleotides
di deoxy nucleotides
are

are
labelled

similar to
w/ a

regular
diff .
colour

( deoxy nucleotides )
of dye
,
but lack a
hydroxyl group
once a di
deoxy nucleotide has been added to the chain there is
hydroxyl available &
°
no
,

no further nucleotides can be added

the nucleotide colour of

chain ends w/ di
deoxy which is marked w/ particular dye depending
•
a on
,

the base ( AT LG ) tht it carries .

method

I .
divide DNA
fragments betw . 4 test tubes
brackets the area for replication & initiates
2 add all 4 nucleotides &
.

primer the replication of DNA by DNA polymerase

3 . to each tube ,
add a diff .
di
deoxy nucleotide tht has been labelled w/ diff . colour dye .

will at
they stop replication at the pt . which
they are incorporated into the DNA strand .

4 let replication far it I repeated fragments of

DNA cycles every possible length
as as can >
go
.

5 .
separate the fragments by gel electrophoresis ( smaller fragments will travel further in the
gel )
6 .

expose get to detector .

the radioactively labelled
dideoxy nucleotide will be detected .

7.
sequence of bases can be read in the chromatogram .
 APPLICATIONS

•
show whether will develop inherited diseases
a
person
detect
changed alleles
by DNA
•

comparing sequences
°

readily identifies :

point mutations
-

small insertions & deletions

other diseases tht can be determined by DNA

sequencing
:

sickle -
cell anaemia

cystic fibrosis
-

some forms of cancer

significance :
ppl who know
they hv inherited a
faulty
allele can seek effective treatment , possibly preventing
the disease OR
reducing its effects
•
other applications :
maternity / paternity tests

4. RECOMBINANT DNA TECHNOLOGY

recombinant DNA ( rDNA) :

organisms / frm orgs

frm
genetic material ( DNA ) tht contains genes / genetic info multiple of
•
.
.

diff species
.

synthetic made frm into DNA frm diff

DNA ;
by inserting genes one source molecules a source
•
.

results in TRANSGENIC ( org tht has material frm multiple

↳
organism .

genetic sources

recombinant DNA ( rDNA) technology

:

used to
procedures produce rDNA
•

involves
introducing DNA into a cell frm diff type of org / DNA tht has been modified in some
•

a .
.

way
aka
genetic engineering
•

tht had frm another introduced into it

artificially
transgenic has DNA
•

' '
org . an
org . .

INGREDIENTS

l . DNA molecules

of interest
gene
•

a
vector

2 .

enzymes
•
restriction enzymes
DNA
ligase
•
vector
bacterial plasmid viral other such
a
phage or
•

agent used to transfer genetic material frm

one cell to another

plasmid limited no ,
of reorg
sites (
.

genes
less likely to impact bacteria life cycle
,
OR

easier to restrict plasmid at specific locations

•
small ,
circular ( strands) of DNA
harder to unzip into strands
.

normally present in bacterial cell ( yeast

normally confer some selective adv to the survival

-

hv one site
ideally only recog
Of
.

bacteria
•

composed of only a few genes & able to ( of chromosomal

replicate independently transformed DNA ) within cells
↳ one bacteria can hv plasmids
many
restriction ( DNA scissors)
enzyme
proteins made
by bacteria
•

of of OR
°

enzyme tht cuts strands DNA at a

specific sequence nucleotides ( bases )

tht catalyse hydrolysis of DNA I cut the phosphodiester bonds in a DNA molecule
enzyme
°

specific sequence of nucleotides ( bases) where the cuts a strand of DNA known
enzyme is

as a
recognition site
↳ at specific restriction / recognition sites (
usually palindromic )
to create ends ( of nucleotides of strand )
↳
sticky overhangs a
single
of infecting virus viral DNA
discovered in bacteria tht restrict duplication by cutting up
°

can

some enzymes produce straight cuts ,

while others produce staggered cuts

straight out

when the
cut
produced RE makes break 2
•

a a a clean across

strands of DNA so tht the ends terminate in a base pair =

blunt ends

staggered out

a cut produced when RE creates fragments of DNA w/ unpaired nucleotides tht

overhang
°

at the break the strands ends

in
sticky
=

sticky ends to combine w/ sections of tht hv

are able DNA a
complementary ending
•

°
a
single -
stranded overhang frm one
fragment can be
paired w/ any other fragment of DNA

tht has a
corresponding sequence
multiply

BB•z*aa
5 treat viruses to take up

treat bacterial
take

pisasmtiod
" up

on
 DNA ligase ( DNA
glue )
of components of single strand
an
enzyme capable combining 2 small DNA into one
•
-

single structure
•

catalyses formation of phosphodiester bonds betw . DNA strands / DNA fragments

'

the phosphate backbone

'
↳ seals nicks in
sugar
-

ligation of
joining short strands of DNA
during replication
°

process

of use of rDNA tech

e.g . .

medical treatment insulin human hormone , factor vill follicle

stimulating hormone ( FSH)
growth vaccines
: -

, , ,

insulin

.
used to treat type I diabetes
•

previously derived frm pancreas of pigs & cattle

°
insulin produced using rDNA technology is identical to human insulin

gene engineered
↳ human into bacteria

reduced side effects suffered frm cattlepigs

by some ppl
•

adv :
when insulin was used

human
growth hormone ( HGH )
•
used to treat children born w/ a form of dwarfism caused by inadequate amnts of HGH
factor vill

used to treat ( or classic haemophilia)

ppl w/ haemophilia A
•

Haemophilia A an inherited disorder in which a blood -

clotting protein known as factor vill is

missing or in poor supply

obtained frm human plasma
previously
°

↳
plasma frm thousands of donors required
↳ risk of transmission of viral disease HIV , hepatitis C

advantages free frm other proteins could I reach

plasma tht cause immune resp allergic
°
: .
.

vaccines
I

hepatitis B vaccine
•

↳
gene frm hepatitis B virus inserted into cowpox virus

•
dis .
of developing rDNA vaccines :

very expensive
-

if a conventional vaccine is known to be safe ,

there is little incentive to develop a new one

using g. e .

5. IDENTIFICATION OF HEREDITARY DISEASES

hereditary diseases
•

state of ill health frm individual 's

a
resulting genes
-

an

caused by defective genetic information

being transmitted frm parents to their children
-

be caused mutations
can
by
-

mutation to characteristic
a chg in a
gene I chromosome
leading a new in an
org .

°
advances in biotech . hv facilitated the development of techniques to identify diseases caused by
mutant
genes

°
short of DNA CRNA )
sequence or

SINGLE stranded
•

COMPLEMENTARY to of
. artificial short sequences
•

synthetic & contains a sequence known sequence DNA

labelled w/ a fluorescent tag OR w/ a radioactive isotope

isotope / dye
.

radioactive
° which are
tagged w/ a

tht has of interest

o a
complementary seq .
to DNA
. bind in a
complementary manner to the target seq .
I known seq / .

complementary seq .

if
•
tagged w/ dye , view under UV
light if
if
°
allows researches to
identify tht
target seq exists in a particular cell / band of a
tagged w/
.

radioactive isotope view under

> X
Ray
• -

genetic profile
 6 . GENE THERAPY CULTURE IN LAB / RECOMBINANT PLASMID

treatment of replacing manipulating supplementing functional

gene therapy by
°
disease ,
or non -

performing normal I expected function

bells & not a

in tissues
genes
most medicines treat of
conventional symptoms disease
gene therapy correct
underlying cause
by
°

replacing the faulty gene w/ a

healthy one .

current focus on single -

gene disorders
autosomal
fibrosis

)
↳
cystic
recessive

"" "
dnonmaihanth monogenic diseases / disorders
,
Huntington 's disease
autosomal
recessive ↳ sickle -
cell anaemia

in
manipulating the
genetic content of some cells
,
NOT whole
org .

effects usually temporary

Method 1 (using viruses)

Place recombinant plasmid that has the functional gene into
-
recessive DNA tech .
An attenuated virus
Virus must target the cells of interest

method 1
method 2 May cause an immune reaction
Diabetes Beta cells insulin
gene Risk of viruses becoming virulent
mutated
Risk of genes inserted in the wrong loci, may result in severe side
① ① supplementing
functional effects such as tumours/ cancers
/ non -

functional genes
insulin gene

Method 2
Isolate host cells first
non pancreas Culture in lab
virulent
Use ( viruses/ gene guns etc) to deliver functional genes into
the cultured cells
Check and ensure that cells have taken up the correct gene/
no noticeable problems
Reintroduce into host

Not likely to create an immune reaction

RISK FACTORS

The of viral vectors could mean tht the viruses deliver the into the
wrong cells / into the
use
gene wrong
part of the chromosome tumour 1
growth
>
cancer

Less virulent viruses could be used ,

viruses could revert to being a more virulent form
cystic fibrosis °
CF was a logical choice for treatment

using gene therapy

disorder
single gene
-
-

most
severely affected lung relatively
-

to & provide treatment

easy access

to
slow
progress 's
lungs of newborns
-

virtually normal ,
enable
gene therapy to

begin b4 significant lung damage started

to occur

Huntington 's disease

mutations chromosome 4 called IT 15
single gene on
•

in a

•
mutated form of protein called hunting tin
nerve cells in brain damaged
-

&
physical mental emotional changes
-

occasional unintentional
flailing movements of arms &
legs
difficulty making voluntary movements of limbs
-

in

dementia ( loss of
progressive ability to think clearly )
-

cell replacement therapy & tissue

engineering
Stem cells pluripotent undifferentiated cells tht capable of repeated
•

are are

mitotic divisions for long periods of time and ,

given right conditions ,
can

differentiate into specialised cells

↳
inject stem cells into indie .

pluripotent differentiate into type of cell except placenta

↳ stem cells can
any
↳ can divide repeatedly
7. CELL REPLACEMENT THERAPY STEM CELLS

replacement of cells
cell replacement therapy damaged w/ healthy ones
•

potential to be applied in any disorder involving loss of ,

or injury to normal cells

most interest Parkinson 's & Alzheimer 's diseases

in nervous
system
°

pilot studies
using embryonic stems in humans carried out w/ some success
,
but raises ethical questions

for Parkinson 's ,

stem cells used to
grow
the
dopamine -

producing nerve cells in

laboratory .

8. TISSUE ENGINEERING

the rebuilding of damaged tissue of &

by the
biology medicine
engineering
•

use ,

aim to eliminate the need for tissue 1 transplants

organ
°

stem cells are introduced to on scaffold of natural or synthetic material to produce

grow
°

3 -
dimensional tissue

scaffold then implanted into the the

cell covered is
patient at site where new tissue is
•
-

required
stem cells outside body hope it into correct tissue then deliver tissue s
indu
grow ,
grows ,
.

tissue
implant

correct
shape