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L20

Selection Review

Bacteria

with
Bacteria
Bacteria

recombinant
without
with plasmid
plasmid (&
plasmid
no fragment
fragment)

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Cut DNA & vector - inserts in every vector ?



Transform bacteria- vectors in every cell ?

Fig 15.6

(a)A vector should be the right size to carry the DNA fragment

(b) there are (engineered) restriction sites in the vector and the
fragment

(c) there are engineered selectable phenotypic characters,

(d) an origin of replication

(e) An easy way to insert into a host,

At this point you can create a LIBRARY of genomic fragments
(of many kinds), stored in vectors, each within different host
cells.

(f) To obtain the clone you want:

(1) direct selection

(2) You can ID a clone from the library with probes .
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Direct selection among recombinant bacterial colonies

(a)Functional dominance test: the host bacteria has a mutation (phe-)
that a specific gene on the recombinant insert (phe+) can rescue.

(b)Functional expression test: can you get an expected, functional protein
expressed - has an intact functional foreign gene been inserted?

If the goal is expressing a eukaryotic protein in a bacterial expression
vector the vector should have key transcription & translation
sequences (bacterial promoter, initiation and termination sequences
regulator and operator genes, ribosome binding site, etc. ) and remove
the introns.. Use a cDNA library

(c)Probe a colony with known complementary sequence:

Select for a library of recombinant cells


Bacteria

without

plasmid

1

Bacteria

with plasmid

no fragment

2

Bacteria

with
recombinant
plasmid (&
foreign
fragment)

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Screen the library for


a sequence of interest

Probe to
locate 1
sequence in a
library of
thousands of
recombinant
cells

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Bacteria with
recombinant plasmid
& the fragment has a
gene or sequence of
interest

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Visualizing DNA of Interest Shotgun analysis of a Library


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You can use a combination of



(1) Southern Blots to separate: different sizes of cDNA
or DNA fragments of different sizes



(2)and probes to identify a specific sequence,
associated with a fragment, gene or cDNA in :



(a)among several recombinant clones

(b) within a pool of other DNA

Denaturation in
an alkaline
solution

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or

Southern, (Northern or Western) blot



1. DNA digestion 2.Electrophoresis

3. Denaturation (to single strand DNA)

4. Transfer to membrane

(blotting).

5. Hybridization with a radioactive single
stranded DNA probe.

6. Autoradiography or Florescence

washing

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Do 2 clone fragments have the same sequence or


gene? Southern blot can answer whether clone x
the same as 2-5.

1. Make southern

blot with clones 1-5.

e.g Clone 4

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32P

Clone x

Probe.

Autoradiograph

Restriction fragment length polymorphism (RFLP)



BRCA1

6 kb

Allele 1


Pst I

Pst I

BRCA1

Allele 2

8 kb

Pst I

Southern

blot of

DNA digested

with Pst I

from 4 persons.

Probed with

BRAC

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Pst I

MW

Autoradiograph

A2

A1

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Southern Blot probed with BRCA1



Restriction Fragment Length Polymorphism (RFLP)

Autoradiograph

If there is a 6 kb band of
DNA

it means that BRCA1 is
flanked by restriction sites
that are 6 kb apart.



This tags or marks a
possible gene polymorphism

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Southern Blot probed with BRCA1



Restriction Fragment Length Polymorphism (RFLP)

RFLP can be used as allelic markers

- linked disease markers.





E.g. If a grandmother who had breast
cancer, had a 8 kb band on a fragment
that cosegregates with a gene that was
known to have a mutation, a
granddaughter could be

checked to test if she inherited

the fragment and presumably the
mutation, unless...

(1) back mutation (2) crossover and
recombination

Autoradiograph

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Some restriction sites mark disease-causing


mutations in a gene, rather than polymorphic
(length) fragments that tag linked gene
polymorphisms (markers have a probability
associated with them ( recombination, mutation,
possibly incidence in a population or pedigree)

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There are different kinds of DNA sequence


DNA can be classified into several different kinds of


sequence by their re-association profile

DNA was sheared into ~ 1000bp long
fragments, then denatured by heating into
single strands. The strands are allowed to reassociate according to their complementary
base pairs under stringent conditions.

Cot - double stranded DNA concentration


in solution (Mol / l) * time.

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Unique sequence DNA - in a haploid genome there are many


genes with a single or few copies.



Middle repetitive DNA -some gene families have multiple
copies and variants, thus a specific probe may detect many
bands (100 -500).



Highly repetitive simple sequence or satellite DNA - some
satellite DNA is repetitive in sequence but hyper-variable, in the
number of simple sequence repeats.



These short sequence repeats are used to:



(a) indicate identity or, identify individual differences through
DNA finger printing.



(b) can be used, as biomarkers, to mark disease alleles, that is,
locate them on a chromosome, in a particular family or lineage.
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DNA fingerprinting is a technique that uses highly repetitive,


satellite DNA to identify individuals and test family relationships
Satellite DNA is often used to lump different kinds of repetitive sequences.



VNTR (Variable Number Tandem Repeats) (1) minisatellites (15-100 base
sequence or unit repeats), microsatellites (or Simple Sequence Repeat or
Short Tandem Repeat DNA (less than 15 -20 unit repeats).



Some are found in euchromatin, scattered throughout the chromosome.

Some tandem repeats are found in large clusters near centromeres, telomeres
suggesting it has a role in meiosis and mitosis, but it is widely found in the
Y chromosome and in heterochromatin where it has no known function
and it is not transcribed.



If restriction enzymes cut sequences that flank VNTR (but not inside), then
the fragment size depends on the number of repeats between the flanking
sequence.

In this case alleles are fragments of different -sized DNA, cut by
restriction enzymes. These can be probed for visualization.


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DNA finger print by


southern blot.

This is the result from
a single probe.

By using several
independent probes
irrefutable data

- evidence - can be

gathered.

Difference- 1 band

Identity - multiple

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Match bands (identity), check inheritance, then estimate the probability


of a match. First estimate the genotype frequency in the population, then
assuming statistical independence of the loci, the frequency of the
multilocus genotype is calculated as the product of genotype frequencies
at each locus (independence rule).


For example, the probability that Dollys genotype


came from another Finn dorset sheep at the Hannah
Research Institute was 5 * 10-11 . In the case of Dolly,
this is strong evidence that her genotype is rare, but
identical to one other individual - her mother.

Establishing
identity
requires:

(1) Multiple loci
(13) agreement

(2) It is always in
the context of
one
populationnot across
populations

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Methods of rapidly generating high DNA copy number,


required for these kinds of analyses.without cloning
fragments and inserting them in a vector maintained in
a bacterial lineage.

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Complementary DNA - a double


stranded version of an mRNA
molecule. It is generated by reversetranscribing DNA from an mRNA
template (using reverse-transcriptase).


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Cells from an organ, tissue,


specific stage of development etc.

AAAA

AAAA

Lyse the cells, extract the RNA,
AAAA



clean it up, add Oligo dT



AAAA


and oligo dT primer

(1)Add reverse transcriptase
AAAA3
mRNA cDNA

TTTT 5
heteroduplex

2 RNA fragments act as primers

(2) Add RNAase (H) nicks & degrades


RNA and may prime synthesis

TTTT5
Single stranded
cDNA

(3) Add DNA polymerase I, then DNA ligase



AAAA3

TTTT 5

(4) Ligate oligonucleotide (T4 ligase) linkers containing EcoR1 for example
and then ligate into a vector

AAAA3 NNGAATTCNN

NNGAATTCNN

TTTT 5 NNCTTAAGNN

NNCTTAAGNN

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cDNA libraries


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Advantages:(1) deciphering the coding region of a gene, since the


structure of a gene is not always obvious from the genomic sequence.


(2) no repetitive sequence which may be a large part of a gene.


(3) eukaryotic cDNA can be expressed in bacteria,
eukaryotic genomic cannot (introns).

(4) construct DNA probes or make a large amount of
DNA from small concentrations of mRNA using PCR (reverse
transcriptase PCR).



Disadvantages (a) a cDNA library is tissue specific it contains only
sequences expressed in the tissue which they are from.

(b) there may not be a full length coding sequence, it
is potentially misleading.

(c) it contains only sequences present in mature DNA,
introns , promoters, (proximal) activators and enhancers are not
present.




We can amplify or make large amount of a sequence of


interest by using a genomic or cDNA library and:



(1)cloning a probed fragment, but, the cells have to be lysed, the
fragments recovered and even then, the gene may not be complete
or it is part of a larger fragment - requiring further processing.



(2)We could amplify large amounts of probed cDNA, in a
plasmid, but the genes may not be complete, promoters and
enhancers need to be engineered.



Or, we could use the probe sequence to design primers for

Polymerase Chain Reaction (PCR). It can amplify specific DNA
sequence (2-35,000 kb) that is initially in low copy number.



But you need to know at least a partial sequence



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PCR Method - pp731 - 732


Denaturation

Heat to 92-95oC for 30s-1min

Annealing - primer specificity



Tm=(4*#G+C)+(2*#A+T) oC

Cool to 2oC below Tm (50-60 oC).

Add excess synthetic primers
for 30s - 1 min

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Extention Heat to 74 C
(Taq optimum).

Add DNA polymerase
Taq, polymerization at
for 90s - 120 sec

Variable length strand


Second cycle

Heat to 92-95oC
for 30s-1min

Cool to 50-60 oC.

Add excess synthetic
primers for 30s - 1 min


Heat to 74 C (Taq
optimum).

Add DNA
polymerase Taq,
polymerization at
for 90s - 120 sec


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Fourth, fifth , etc



produce an exponential

increase in copy number

Third cycle

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Link a restriction sequence


to the primer - insertion
ready

Primer 1-HindIII

EcoR1-Primer 2

With a partially known sequence you can design


primers to:

Amplify cDNA(reverse transcriptase PCR) and DNA

Probe for genes on a fragment and much more..


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Using satellite DNA


variation to mark disease related sequence

From the PCR products

1)Which locus- disease

2)Dominant or recessive,

Sex linked or autosomal ?

Why clone a gene, why not use cDNA, or PCR ?




cDNA can be used to copy a gene, with a little engineering, a


eukaryotic gene can be expressed in bacteria.

But cDNA is often incomplete, lacking 5 end sequence.



A DNA molecule is copied during PCR between the annealing
positions of 2 oligonucleotide primers. A PCR experiment can
be finished in a few hours, whereas cloning may take weeks
or months.

But : (1) multiple gene splicing

2) PCR cannot be used to isolate genes that have not been
studied before- they have to be cloned or at least partially
sequenced, to use the technique (primer sequences).

3) PCR may have a high error rate - importance of annealing

4) PCR has a limited number of base pairs that it can copy: 4 Kb
up to 40Kb- which is shorter than most genes- if an intact
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version is required, longer genes must be cloned first.