L18Biol261 DNA TechnolWRecomb

8/9/2019 L18Biol261 DNA TechnolWRecomb

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L20


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Bacteriawithout

plasmid

Bacteriawith plasmidno fragment

Bacteria

withrecombinant

plasmid (&fragment)

1 2

3

Selection Review

Cut DNA & vector- inserts in every vector?Transform bacteria-vectors in every cell?

Fig 15.6

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

(b) there are (engineered) restriction sitesin the vector and the

fragment

(c) there are engineered selectable phenotypic characters,

(d) an origin of replication

(e) An easy way toinsertinto a host,

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

(f) To obtain the clone you want:(1) direct selection

(2) You can ID a clone from the library with probes . 5


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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)Functionalexpressiontest: can you get an expected, functional protein

expressed - has an intact functional foreign gene been inserted?

If the goal isexpressing a eukaryotic proteinin a bacterialexpression

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:

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Bacteria

without

plasmid

Bacteriawith plasmidno fragment

Bacteriawithrecombinant

plasmid (&foreignfragment)

1 2

3

Bacteria withrecombinant plasmid& the fragment has a

gene or sequence ofinterest

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Select for a library of recombinant cells

Probe tolocate 1sequence in alibrary ofthousands of

recombinantcells

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Screen the library fora sequence of interest


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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

11Visualizing DNA of Interest -

Shotgun analysis of a Library


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Denaturation inan alkalinesolution

washing

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Southern, (Northern or Western) blot1. DNA digestion 2.Electrophoresis3. Denaturation (to single strand DNA)

4. Transfer to membrane(blotting).5. Hybridization with a radioactive single

stranded DNA probe.6. Autoradiography or Florescence

1

4

3 or


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Do 2 clone fragments have the same sequence orgene? Southern blot can answer whether clone xthe same as 2-5.

e.g Clone 4

1. Make southernblot with clones 1-5. Autoradiograph

Clone xProbe.32P

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Restriction fragment length polymorphism (RFLP)

Allele 1

BRCA1

Pst I Pst I

Allele 2BRCA1

Pst I Pst I

8 kb

6 kb

AutoradiographSouthernblot ofDNA digested

with Pst Ifrom 4 persons.

Probed withBRAC

MW 1 2 3 4

A2

A1

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Southern Blot probed with BRCA1Restriction Fragment Length Polymorphism (RFLP)

Autoradiograph

If there is a 6 kb band of

DNAit means that BRCA1 is

flanked by restriction sitesthat are 6 kb apart.

This tags or marks apossible gene polymorphism

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Southern Blot probed with BRCA1Restriction Fragment Length Polymorphism (RFLP)

AutoradiographRFLP can be used as allelic markers- linked disease markers.

E.g. If a grandmother who had breastcancer, had a 8 kb band on a fragmentthat cosegregates with a gene that wasknown to have a mutation, agranddaughter could be

checked to test if she inheritedthe fragment and presumably themutation, unless...(1) back mutation(2) crossover andrecombination

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Some restriction sites mark disease-causingmutations in a gene, rather than polymorphic

(length) fragments that tag linked genepolymorphisms (markers have a probabilityassociated with them ( recombination, mutation,

possibly incidence in a population or pedigree)

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There are different kinds of DNA sequence


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DNA can be classified into several different kinds ofsequence by their re-association profile

Cot - double stranded DNA concentrationin solution (Mol / l) * time.

DNA was sheared into ~ 1000bp longfragments, then denatured by heating intosingle strands. The strands are allowed to re-associate according to their complementarybase pairs under stringent conditions.

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Unique sequenceDNA - in a haploid genome there are manygenes with a single or few copies.

Middle repetitiveDNA -some gene families have multiplecopies and variants, thus a specific probe may detect manybands (100 -500).

Highly repetitivesimple sequence orsatellite DNA- somesatellite DNA is repetitive in sequence but hyper-variable, in thenumber of simple sequence repeats.

These short sequence repeats are used to:

(a) indicate identity or, identify individual differencesthroughDNA 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 DNAis often used to lump different kinds of repetitive sequences.

VNTR (Variable Number Tandem Repeats) (1) minisatellites(15-100 basesequence or unit repeats), microsatellites(or Simple Sequence Repeat orShort 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, telomeressuggesting it has a role in meiosis and mitosis, but it is widely found in theY chromosome and in heterochromatin where it has no known functionand it is not transcribed.

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

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 bysouthern blot.This is the result froma single probe.By using severalindependent probesirrefutable 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 independenceof the loci, the frequency of the

multilocus genotype is calculated as the product of genotype frequenciesat 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.

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Establishingidentityrequires:

(1)

Multiple loci(13) agreement

(2)It is always inthe context ofone

population-not acrosspopulations


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Methods of rapidlygenerating high DNA copy number,required for these kinds of analyses.without cloningfragments and inserting them in a vector maintained ina bacterial lineage.

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Complementary DNA- a doublestranded version of an mRNAmolecule. It is generated by

reverse-transcribingDNA from an mRNAtemplate (using reverse-transcriptase).

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Cells from an organ, tissue,specific stage of development etc.Lyse the cells, extract the RNA,clean it up, add Oligo dT

AAAAAAAA

AAAAAAAA

(1)Add reverse transcriptase and oligo dT primer

AAAA3

TTTT5

mRNAcDNAheteroduplex

(2) Add RNAase(H)nicks & degradesRNA and mayprime synthesisTTTT5

Single strandedcDNA

(3) Add DNA polymerase I, then DNA ligaseAAAA3

TTTT 5

AAAA3

NNGAATTCNNTTTT 5

NNCTTAAGNN

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

NNCTTAAGNNNNGAATTCNN

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2

RNA fragments act as primers


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cDNA libraries

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

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

(2) no repetitive sequencewhich 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 (reversetranscriptase PCR).

Disadvantages (a) a cDNA libraryis tissue specificit contains onlysequences expressed in the tissue which they are from.

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

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

present.

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We can amplify or make large amount of a sequence ofinterest by using a genomic or cDNA library and:

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

(2)

We could amplify large amounts of probed cDNA, in a

plasmid,butthe genes may not be complete, promoters andenhancers need to be engineered.

Or, we could use the probe sequence to design primers forPolymerase 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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ExtentionHeat to 74 C(Taq optimum).

Add DNA polymeraseTaq, polymerization atfor 90s- 120 sec

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

Annealing- primer specificity

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

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

Add excess synthetic primersfor 30s - 1 min

PCR Method - pp731 - 732


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Second cycle

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Variable length strand

Heat to 92-95oCfor 30s-1min

Cool to 50-60 oC.Add excess synthetic

primers for 30s - 1 min

Heat to 74 C (Taqoptimum).

Add DNApolymerase Taq,polymerization atfor 90s- 120 sec


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Third cycle

Fourth, fifth , etcproduce an exponential

increase in copy number

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Primer 1-HindIII

EcoR1-Primer 2

Link a restriction sequenceto the primer- insertion

ready

With a partially known sequence you can designprimers to:

Amplify cDNA(reverse transcriptase PCR)and DNAProbe for genes on a fragment and much more.. 35


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Using satellite DNAvariation to mark disease -

related sequence

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1)Which locus- disease

2)

Dominant or recessive,Sex linked or autosomal ?

From the PCR products


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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 annealingpositions of 2 oligonucleotide primers. A PCR experiment can

be finished in a few hours, whereas cloning may take weeksor months.

But : (1) multiple gene splicing2) PCR cannot be used to isolate genes that have not beenstudied 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 annealing4) PCR has a limited number of base pairs that it can copy: 4 Kbup to 40Kb- which is shorter than most genes- if an intactversion is required, longer genes must be cloned first. 37

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L18Biol261 DNA TechnolWRecomb