Recombinant DNA

Restriction enzymes
Restriction mapping
DNA cloning
Libraries
Methods of detection
Bam HI non-specific site
Bam HI specific site

http://www.biochem.arizona.edu/classes/bioc471/pages/Lecture2/Lecture2.html0
Restriction/modification systems
http://www.microbelibrary.org/microbeli
brary/files/ccImages/Articleimages/Atlas_
Plaque%20Assay/Figure%201%20Pages%2
01%203.jpg
E. coli B
E. coli K12
2
http://www.scq.ubc.ca/restriction-endonucleases-molecular-scissors-for-specifically-cutting-dna/
Restriction/modification systems
See Supplement p. 64, bottom
Restriction Enzymes: Restrict biological

activity/growth of viruses
Modification Enzymes: Methyl transferases.
Methylate DNA to protect from cleavage
Both recognize the same palindromic
recognition sequence
Figure p. 293
Hartwell, Genetics: From
Genes to Genomes, 4e
Restriction enzymes are endonucleases

(type II)
6 bp
sites
4 bp
sites
Legos
= axis of symmetry
= site of strand cleavage
http://barleyworld.org/css430_09/lecture%208-09/figure-11-04.JPG
What does dyad symmetry of cleavage site indicate

about the active form of restriction enzymes?
Restriction Enzymes function as homodimers
http://www.bpc.mh-hannover.de/alves/Gruppe_Alves_engl.html
Nice animation at
http://www.youtube.com/watch?v=aA5fyWJh5S0
arbeitsweise = function
erkennung = recognition
spaltung = splitting
The calculation shown assumes:

1) Genome composition is 50% (G + C)
2) Nucleotides are distributed randomly throughout genome
NOTE: Restriction sites are not actually evenly distributed along DNA!
Figure 9.3a
Agarose gel electrophoresis
7
http://www.phschool.com/science/biology_place/biocoach/red/gel.html
Distance migrated is
influenced by size of
DNA and percentage
of agarose in gel
HSE-SP6
http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html
Banding pattern is specific

to the DNA sample
Log (size) is inversely proportional

to distance migrated (X).
W C100
lCI
3 kb
2 kb
0.7 kb
Photo: MF Gaudette
Restriction Mapping
See Supplement p 72
The undigested linear DNA = 10 kb

EcoRI cuts the DNA twice
HindIII cuts the DNA once
Double digest yields 2 new fragments:
4 kb and 1 kb
Indicates the HindIII site is within the
5 kb EcoRI fragment
Indicates that the 5 kb EcoRI fragment is the

internal fragment
The 3 kb and 2 kb EcoRI fragments are at
either end
For an additional mapping problems (with

solutions), see Supplement p 73-74
9
10
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=hmg&part=A359&rendertype=figure&id=A361
Vectors need
an origin of replication compatible with host cell replication enzymes
a convenient site for insertion of foreign DNA
a route of entry into the host cell
a means to identify cells that have taken up the vector
a means to identify cells that have taken up a recombinant vector
TABLE 9.2
11
Creating recombinant DNA molecules
*
Notice duplication
of restriction
enzyme
recognition site
*or one that

generates
compatible
ends
Origin of DNA
does not
influence ability
to base-pair
12
http://campus.queens.edu/faculty/jannr/Genetics/images/dnatech/bx15_01.jpg
Cloning in a plasmid
See Supplement p 75
http://delliss.people.cofc.edu/virtuallabbook/DrugRes/AntibioticRes.html
Transform antibioticS
bacterial cells
plated
Selection
Only bacteria that have incorporated the

plasmid (with the antibioticR gene) will survive
But how do we identify the cells that have taken up

plasmids that contains foreign DNA?
13
REVISED from http://www.accessexcellence.org/RC/VL/GG/plasmid.php
Interrupt a gene that has

a detectable phenotype
Plate on Medium + Amp (Selection)
Selects for ampicillin-resistant cells, which
have incorporated plasmid molecules
Does not distinguish between TetR and TetS
<
Replica plate on Medium + Tet (Screening)

Selects for tetracyclin-resistant cells, which
have not incorporated recombinant
plasmids
Go back to master plate & Pick colonies that grow
on Med + Amp but not on Med + Tet
14
MCS = multiple cloning site
(= MCS)
15
REVISED from http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2027.jpg
http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=B3928|SIGMA&N5=SEARCH_CO
NCAT_PNO|BRAND_KEY&F=SPEC&lang=en_US%3E#
Beta-galactosidase converts colorless

X-Gal to a blue insoluble compound
D
<
All colonies are antibiotic resistant (Selection)

Blue colonies have incorporated plasmid with no insert
(Screening)
lacZ is intact
White colonies have incorporated recombinant plasmid
Foreign DNA insert interrupts and inactivates lacZ
16
Cloning in a lambda
Remember that
phage can only
carry one headful
of DNA.
Some phage gene
sequences need to
be retained for
successful lysis.
17
Cloning in a BAC
18
http://www.scq.ubc.ca/the-big-bad-bac-bacterial-artificial-chromosomes/
Cloning in a YAC
19
http://themedicalbiochemistrypage.org/molecular-medicine.html
20
Constructing a genomic library
Genome equivalent =
Haploid genome size
divided by average
insert size
Gives the minimum
number of clones
needed to be
screened to identify
single-copy genes
Figure 16.7 PJ Russell iGenetics, A Mendelian Approach AND

21
Constructing a cDNA library
AND http://www.mun.ca/biology/desmid/brian/BIOL2060/BIOL2060-20/2030.jpg
22
Figure 9.9
23
Expression vectors
http://www.bio.davidson.edu/courses/genomics/method/plasmid_inducible.html
Figure 1. Map of a generic expression

vector. This simplistic example contains an
inducible promoter (red), your favorite
gene (green), a gene that confers
antibiotic resistance (blue), and an origin
of replication (orange) used to produce
many copies of the plasmid.
http://wolfson.huji.ac.il/expression/vector/vec-anat.html
24
25
Hybridization
NOTE: This figure shows annealing of

target and probe molecules in solution
Figure 5.8
A nucleic acid hybridization assay requires the formation of heteroduplexes between labeled
single-stranded nucleic acid probes and complementary sequences within a target nucleic acid
26
Hybridization probes
27
Hybridization
Figure 5.10 Nucleic acid hybridization can identify target sequences

that are considerably diverged from a conventional DNA (or RNA)
probe, or that are identical to an oligonucleotide probe
28
Colony Hybridization/detection
(Treat with alkali

+ protease)
Block membrane to prevent

non-specific sticking of probe
to membrane
29
Stringency high or low will

affect the level of probe binding
30
Figure 16.16
PJ Russell iGenetics, A Mendelian Approach
Southern Blot Analysis
Block membrane to
prevent non-specific
sticking of probe to
membrane
wash
Denature and
31
http://www.scq.ubc.ca/dna-fingerprinting-in-the-standardization-of-herbs-and-nutraceuticals/
Southern blot analysis of total Arabidopsis DNA at high

and low stringency using 32P-labeled GMD2 as a probe
Southern blots probed with GMD2.
A. High stringency conditions
A single hybridizing fragment is observed
B. Reduced stringency conditions
Two fragments are observed
This result provides further evidence that
two genes for putative
GDP-D-mannose-4,6-dehydratases are
present in the Arabidopsis genome.
High stringency
Bonin C P et al. PNAS 1997;94:2085-2090
1997 by The National Academy of Sciences of the USA
Reduced stringency
High stringency = 68C hybridization,

65C washes.
Low stringency = 58C hybridization,
55C washes
32
A zoo blot
Reduced stringency
conditions allow
greater mismatches
in probe-sequence
hybridizations
allows identification
of sequences that
are similar but not
necessarily identical
VDAC1 = Voltage-dependent
anion-selective channel
protein 1
33
MJ Sampson et al., (1997 ) J Biol. Chem. 272 (30): 1896618973,
Northern Blot Analysis
06_13.jpg
mRNA from various tissues is

separated in a denaturing gel and
probed for a specific sequence.
Here, the probe is labeled cDNA from

the FMR1 (fragile X mental retardation
1) gene.
What question is being asked?
The protein is made in many
tissues, but at high levels in the
brain & testes.
34
Western Blot Analysis
http://visiscience.com/samples/methods/Far-Western-Blotting.jpg
http://www.lookfordiagnosis.com/mesh_info.php?term=Western+Blotting&lang=3
35
Western Blot Analysis
Run samples on PAGE gel

Transfer proteins to
nitrocellulose membrane
Block membrane
Prevents non-specific
adsorption of antibody
Incubate with primary

antibody
Binds target protein(s)
Incubate with 2 antibody

Binds 1 antibody
Carries enzyme allows detection
Wash
Expose blot to chromogenic
substrate
Detect color
= Site of antibody binding
Therefore site of target protein
Wash
36
http://biotech.matcmadison.edu/resources/proteins/labManual/chapter_5/procedure5_4.htm
37
Polymerase Chain Reaction (PCR)

Amplifies DNA quickly
Provides DNA for additional
investigation
Can be accomplished with
very small amounts of
input DNA
Limited sequence
knowledge is needed
Is the basis for many
diagnostic and forensic
DNA procedures
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=genomes.figgrp.5996
MODIFIED from:
http://www.bioedonline.org/slides/slide01.cfm?tk=44&dpg=11
http://www.emunix.emich.edu/~rwinning/genetics/tech2.htm
Figure 6.2. PCR primer design
39
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=hmg&part=A551
Lower case letters = coding strand of the SRY gene

Upper case letters = primers used to amplify entire SRY gene
(NOTE: forward primer anneals to template DNA strand not shown)
5gttgaggggg
GTTGAGGGGG
5
gaatctggta
ttttgtcgca
Attcaacagc
ctcttccttc
cagtaaaggc
tcgcgatcag
caagcagctg
Ggaggcacag
Tcgtcggaag
ggtactctgc
cacacactca
Accgcagcaa
ggctacaaag
tgttgagggc
TGTTGAGGGC
ggagaaatgc
aagtttcatt
acaaaagtta
acgtaacaaa
ttggatagta
ttttgacaat
gtccagctgt
aaagctgtaa
atagagtgaa
tggctctaga
ggaaaatgct
ccatgcacag
tgccgaagaa
aactggacaa
accagctagg
acagccactg
atgctccttt
aaataagttt
gcaatcatat
gcaagagaat
ctctaagtat
gcgacccatg
gaatcccaga
tactgaagcc
agagaaatac
ttgcagtttg
caggttgtac
ccacttaccg
gacaaagctg
ttacgataac
cgaactctgg
gcttctgcta
attcccgctc
cagtgtgaaa
aacgcattca
atgcgaaact
gaaaaatggc
ccgaattata
cttcccgcag
agggatgact
cccatcaacg
taggacaatc
ttacagccct
cacctttcaa
tgttaagcgt
tccggagaag
cgggagaaaa
tcgtgtggtc
cagagatcag
cattcttcca
agtatcgacc
atcccgcttc
gtacgaaagc
cagccagctc
gggtaacatt
cactttctta
3
gaagtgagtt
ctctccttgt
gatgattaca
ctttgcactg
aacgtccagg
aggcgcaaga
ggataccagt
aaattacagg
gcgaagatgc
agcgaagtgc
agaatggagc
cgggaccgct
acctacctag
3
tgtttagttt
caatattgtt
ttcttttctc
ATT
tggctaataa
TCCGGAATAA
aggccttatt
Some knowledge of the gene sequence is needed

how much depends on your goal.
GTAAAGT
catttca
3
40
Usually 20-30 cycles
41
42
See Supplement p 79-80
Theoretical amplification assumes 100% efficiency
Figure 9.12.4
AMOUNT OF DNA
http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=hmg&part=A551
10000000000
1000000000
100000000
10000000
1000000
100000
10000
1000
100
10
1
0
10
15
20
25
30
PCR CYCLE NUMBER
43
35
Figure 6.4. PCR has numerous general applications
44
Cloning of PCR products
45
Sequences other than

restriction recognition
sites can also be used.
For example,
sequences known to
be homologous to
chromosomal regions
are useful.
46
Disadvantages of cloning by PCR amplification

Small size of amplified product
Although long PCR has been developed, using more
processive polymerases, results are usually mixed
Need for some target sequence information
Cloning of DNA cleaved by restriction digests requires no
prior knowledge of sequence
Low fidelity of replication
Taq DNA polymerase, derived from T. aquaticus, has no
associated 3 5 exonuclease to confer a proofreading
function.
Alternative heat-stable DNA polymerases that have
associated 35 exonuclease activity are currently used.
(eg, the Pyrococcus furiosus (Pfu) polymerase)
47
Figure 6.20. PCR mutagenesis

(B) Site-specific mutagenesis generate an
amplified product with a specific predetermined mutation located in a central
segment.
PCR reactions A and B amplify overlapping

segments of DNA containing an introduced
mutation (by deliberate base mismatching
using a mutant primer - 1M or 2M).
The two products are combined, denatured
and allowed to reanneal. DNA polymerase
can extend the 3 end of heteroduplexes
with recessed 3 ends. Thereafter, a full
length product with the introduced
mutation in a central segment can be
amplified by using the outer primers 1 and
2 only.
48
Real Time PCR
SYBR Green method of qRT-PCR

(quantitative reverse transcription-PCR)
http://pathmicro.med.sc.edu/pcr/SYBRGreen.htm
SYBR green fluoresces only when bound to dsDNA. Quantifying the level
of fluorescence will therefore quantify the amount of amplified product.
But this cannot distinguish between target dsDNA and non-specific
annealing of primers to each other (primer dimers).
49
Real Time PCR
TaqMan assay: one of the earliest methods

introduced for real time PCR reaction
monitoring . Used for:
quantification of mRNAs
detecting variation of expression
Principle: exploits the 5' endonuclease activity
of Taq DNA polymerase, which cleaves an
oligonucleotide probe during PCR, thereby
generating a detectable signal.
Probe: fluorescently labeled at the 5' end and
are non-extendable at their 3' end by chemical
modification.
Specificity is conferred at three levels: via two
PCR primers and the probe.
50
http://www.ncbi.nlm.nih.gov/projects/genome/probe/doc/TechQPCR.shtml
DNA Sequence analysis
See Supplement p 77
Sequencing needs:
ssDNA template
Primer
Polymerase
dNTPs
ddNTPs
Label
often primer is end-labeled
or labeled nucleotides are incorporated
51
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
DNA Sequence analysis
52
http://wiki.biomine.skelleftea.se/biomine/molecular/index_14.htm
Figure 9.13.3
Hartwell, Genetics: From Genes
to Genomes, 4e
53
What sequence is given here?

What is the sequence of the template strand?
This technique is limited by the

resolving capacity of the gel
54
Automated
sequencing
55
Automated
sequencing
Limiting quantities of
fluorescently-labeled ddNTP
T
C
G
T
A
T
G
C
A
A
This technique allows for

sequencing of longer DNA
fragments
56
Longer sequences can be read using automated sequencing
57
http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html
Putting short sequences together to determine sequence of a gene:
See also, supplement p 88 shotgun sequence assembly
58
Pyrosequencing (454 Life Sciences)

Individual DNA molecules are captured on
a bead.
DNA is amplified around the bead.
Beads (covered with DNA) are extracted
and put on a chip with 1.3 million small
wells with a mix of enzymes.
Four nucleotides are washed over in series.
The addition of one or more nucleotides
results in light signal, which is recorded.
Approximately 100 million bases per run.
http://www.bioedonline.org/slides/slide01.cfm?q=pyrosequencing&dpg=1&95009
59
Figure 1 - Diagram of the pyrosequencing process.

From: The development and impact of 454 sequencing
Jonathan M Rothberg & John H Leamon
Nature Biotechnology 26, 1117 - 1124 (2008) Published online: 9 October 2008
The template strand is represented in red, the

annealed primer is shown in black and the DNA
polymerase is shown as the green oval.
Incorporation of the complementary base (the
blue "G") generates inorganic pyrophosphate
(PPi), which is converted to ATP by the
sulfurylase (blue arrow).
Luciferase (red arrow) uses the ATP to convert
luciferin to oxyluciferin, producing light.
http://www.nature.com/nbt/journal/v26/n10/fig_tab/nbt1485_F1.html#figure-title
60
Hall, N. J Exp Biol 2007;210:1518-1525
61

Recombinant DNA

Hochgeladen von

Dokumentinformationen

Originaltitel

Copyright

Verfügbare Formate

Dieses Dokument teilen

Dokument teilen oder einbetten

Freigabeoptionen

Stufen Sie dieses Dokument als nützlich ein?

Sind diese Inhalte unangemessen?

Copyright:

Verfügbare Formate

Recombinant DNA

Hochgeladen von

Copyright:

Verfügbare Formate

Restriction enzymes

Bam HI non-specific site

Bam HI specific site

See Supplement p. 64, bottom

Restriction Enzymes: Restrict biological

Restriction enzymes are endonucleases

What does dyad symmetry of cleavage site indicate

Restriction Enzymes function as homodimers

The calculation shown assumes:

Agarose gel electrophoresis

Banding pattern is specific

Log (size) is inversely proportional

The undigested linear DNA = 10 kb

Indicates that the 5 kb EcoRI fragment is the

For an additional mapping problems (with

Creating recombinant DNA molecules

*or one that

Only bacteria that have incorporated the

But how do we identify the cells that have taken up

Interrupt a gene that has

Replica plate on Medium + Tet (Screening)

MCS = multiple cloning site

Beta-galactosidase converts colorless

All colonies are antibiotic resistant (Selection)

Constructing a genomic library

Figure 16.7 PJ Russell iGenetics, A Mendelian Approach AND

Constructing a cDNA library

Figure 1. Map of a generic expression

NOTE: This figure shows annealing of

Figure 5.10 Nucleic acid hybridization can identify target sequences

(Treat with alkali

Block membrane to prevent

Stringency high or low will

Southern Blot Analysis

Southern blot analysis of total Arabidopsis DNA at high

1997 by The National Academy of Sciences of the USA

High stringency = 68C hybridization,

Northern Blot Analysis

mRNA from various tissues is

Here, the probe is labeled cDNA from

Western Blot Analysis

Western Blot Analysis

Run samples on PAGE gel

Incubate with primary

Incubate with 2 antibody

Polymerase Chain Reaction (PCR)

Figure 6.2. PCR primer design

Lower case letters = coding strand of the SRY gene

Some knowledge of the gene sequence is needed

Usually 20-30 cycles

See Supplement p 79-80

Theoretical amplification assumes 100% efficiency

PCR CYCLE NUMBER

Figure 6.4. PCR has numerous general applications

Cloning of PCR products

Sequences other than

Disadvantages of cloning by PCR amplification

Figure 6.20. PCR mutagenesis

PCR reactions A and B amplify overlapping

Real Time PCR

SYBR Green method of qRT-PCR

Real Time PCR

TaqMan assay: one of the earliest methods