Lecture 1 Isolating DNA Printable Version
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Transcript of Lecture 1 Isolating DNA Printable Version
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CMB2000 – Lecture 1
CMB2000
Principles and Practice of Molecular
Techniques
Dr Geof [email protected]
Telephone 0191 222 5315
Changing the way nature works
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CMB2000 – Lecture 1
CMB2000
Principles and Practice of Molecular
Techniques
Dr Geof [email protected]
Telephone 0191 222 5315
Changing the way nature works
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E.coli: genoe ! " # 10$ %ase pairs &sall'
Isolate and manipulate a defined section of the genome
T(picall(: a gene &coding region for a protein' )00 – )*000 %ase
pairs in length
CMB2000 – Lecture 1
+uan: genoe ! , # 10- %ase pairs &large'
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CMB2000 – Lecture 1
Isolating DNA
tep 1 – release the / fro the cell
• Ph(sical ethods
• Biological ethods
• Mechanical ethods
• Efficient
• ot denature or change the / until e ant to
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• Ph(sical ethods
• 3sotic shoc4
CMB2000 – Lecture 1
Isolating DNA/
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• Ph(sical ethods
• 3sotic shoc4
CMB2000 – Lecture 1
Isolating DNA
tep 1 – release the / fro the cell
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tep 1 – release the / fro the cell
• Ph(sical ethods
CMB2000 – Lecture 1
Isolating DNA
• 5ree6e7tha
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tep 1 – release the / fro the cell
• Biological ethods
•
L(tic en6(es
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tep 1 – release the / fro the cell
• Manual grindersPestle and ortar
+oogeniser
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Isolating DNA10
• Mechanical ethods
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tep 1 – release the / fro the cell
• Blenders
• 8ltrasonic disruptors
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Isolating DNA11
• Mechanical ethods
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Pressure homogenisers
tep 1 – release the / fro the cell
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Isolating DNA13
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tep 1 – release the / fro the cell
CMB2000 – Lecture 1
Isolating DNA
tep 2 – centrifuge don cell fragents at lo rp
tep , – degrade ith ase
tep " – separate the / fro cell proteins ; lipids
DNA extractioncan be done with very
simple ingredients!!
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tep 1 – release the / fro the cell
CMB2000 – Lecture 1
Isolating DNA
tep 2 – centrifuge don cell fragents at lo rp
tep , – degrade ith ase
tep " – separate the / fro cell proteins ; lipids
Phenol
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CMB2000 – Lecture 1
Isolating DNA
7 i# ith equal 9olue ofphenol
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tep 1 – release the / fro the cell
CMB2000 – Lecture 1
tep 2 – centrifuge don cell fragents at lo rp
tep , – degrade ith ase
tep " – separate the / fro cell proteins ; lipids
7 i# ith equal 9olue ofphenol
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Descrie the techni!ue "or isolating D#$
De%ne what &estriction en'onucleasesare an' how they are use' to cut an'
reco(ine D#$
Learning outcomes
CMB2000 – Lecture 1
Isolating DNA
• E9ol9ed %( %acteria to protect against !iral DNA infection
• estriction En6(es cut DNA
- "estriction ! act on specific DNA sequences
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#ndonucleases ! clea9e ithin / strands
De%ne what &estriction en'onucleasesare
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CMB2000 – Lecture 1
Isolating DNA20
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CMB2000 – Lecture 1
Isolating DNA
• E9ol9ed %( %acteria to protect against !iral DNA infection
• estriction En6(es cut DNA
- "estriction ! act on specific DNA sequences
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#ndonucleases ! clea9e ithin / strands• 39er $%000 &no'n en(ymes
• aed for %acterial genus* species* strain and t(pe
E#aple: EcoR I>enus: Escherichia
pecies: coli
train:
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CMB2000 Lecture 1
Isolating DNA
Blunt end )tic&y end
Pallindrome! ord spelled the sae
forards and %ac4ards
eye
racecar
hannah
Pallindrome
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)?* *
* +
Base
*
* *P
,
,?
+**
* +
Base
,?
)?
+2** *
*+*
Base
**
+*
*
* P ,
Base
)?
,?
Restriction
endonuclase
CMB2000 Lecture 1
Isolating DNA
Clea9e at their recognition site through
the phosphodiester %ac4%one in the presence of Mg2-
@ +(drol(ses the phosphate group
@ Cut ends ha9e a )A phosphate
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,se electrophoresis to separate D#$"rag(ents
Learning outcomes
CMB2000 Lecture 1
Isolating DNA2.
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"estriction endonucleases can .e used to cut
DNA into defined fragments/
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Isolating DNA5
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"estriction endonucleases can .e used to cut
DNA into defined fragments/
Isolating DNA/
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"estriction endonucleases can .e used to cut
DNA into defined fragments/
Isolating DNA
+o often ould a $%p restriction endonuclease cut a
hole genoe
3rganis >enoe si6e o. of sitesirus 10) %p 2."
Bacteria 10D %p 2""
Proto6oan 10= %p 2""0
+uan ,#10- %p 1.2#10)
estriction digests of large genoes run as a smear
To ap these requires )outhern .lotting:
transfer / to a e%rane and pro%e for specific
sequence
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Isolating DNA
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Agarose Gel
Electrophoresisells
canning)lectron4icrograph o"$garose Gel161 7(8
+oly(erise' agarose isporous
allowing "or the (o-e(ent o"D#$ acts as a :(olecularsie-e;8
Isolating DNA9
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<
-
Directiono"
D#$ Tra-el
(all
=arge
Agarose Gel
Electrophoresis
Isolating DNA0
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a(ples enter the gel an'(igrate accor'ing to charge
size an' shape>@ DNA is negatively charged -
(igrates to +ve electrode
@ Smaller molecules (o-e (ore
easily through gel than largermolecules
@ Compact molecules (o-e(ore easily through the gel than
longer molecules@ Visualise ith intercalatin
Agarose Gel
Electrophoresis
Isolating DNA1
Pulse field gel electrophoresis
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Isolating DNA
Determine the size of the DNA fragments by comparisonwith the DNA ladder.
)i(e of DNA.and
og10 DNA si(e Distance migratedfrom 'ell mm3
"*000%p
,*000%p
2*000%p
1*$)0%p
1*000%p
=)0%p
$)0%p
)00%p
"00%p
2
Agarose Gel
Electrophoresis
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Isolating DNA
)i(e of DNA.and
og10 DNA si(e Distance migratedfrom 'ell mm3
"*000%p
,*000%p
2*000%p
1*$)0%p
1*000%p
=)0%p
$)0%p
)00%p
"00%p
Then draw a graph
d i s t a n
c e m i g r a t e d m m
log DNA size
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Agarose Gel
ElectrophoresisDetermine the size of the DNA fragments by comparisonwith the DNA ladder.
R d l3.
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>TTCCTT>
>TTCCTT>
>CTT
TTC>
>CTT
TTC>
Cut withEco
RI
DNA ligase! AT"#
$CTTAA
AATTC$
R c mbin nt DNA m l cul
Restriction endonucleases .
