L17Biol261FragInsertDNA2013

8/9/2019 L17Biol261FragInsertDNA2013

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Genetics-deals with problems of heredity and variationon the molecular level

-

Inheritance -Transmission genetics and DNA REPLICATIONWe know how DNA replicates,

interactions between DNA and a host of proteins which open replication forks, unwind, replicate

and eventually twist and stabilize a daughter and parental strand into a molecule of DNA or a

chromosome. We know how chromosomes replicate, how they assort in meiosis and something

about how genes are organized in a chromosome - as a stand alone unit of inheritance,

variation and expression.

VariationMUTATION AND VARIATION - Molecular and VisibleWe know there are alleles

and that genes could mutate. Single base substitutions may cause amino acid and transcript

length changes. Furthermore, chromosome pieces could be inserted, deleted, inverted,

duplicated, translocated and otherwise rearranged. Unless single base or chromosome

fragments are repaired, mutations are replicated in mitosis as cells divide and meiosis as germ

cells replicate. We also know how fragments can migrate throughout the genome, transposed by

several mechanisms: families of cut and paste transposons that move and sometimes take

fragments with them; retrotranspons and transposons that replicate themselves and other

contained fragments and then they delete or insert (indels). These are the mechanisms generating

genetic variation, some silent, nonfunctional or hidden, some with little difference from the wild

or standard phenotype or neutral, some variants associated with significant character changes, for

better or worse, they are tested in the laboratory of life.


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Cutting and Inserting

recombinant DNA


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DNA Technology I: making fragment

libraries and identifying genes Wwill look at how the following,

familiar definitions and topics are applied:

(1) endonucleasesand chromosome cuts

(2) extrachromosomal DNA e.g. plasmidsthat can carry foreign DNA

(3) reverse transcriptaseand cDNA (4) Southern blots and fragment sorting

(5) complementary bindingand probes

(6) plasmids and bacterial chromosomes

OBJECTIVES

To:(a) store and replicatespecific fragments of DNA,

(b) identifyspecific genes, or,(c) expresslarge amounts of protein. 2


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DNA technologyis a term that describes the collective

techniques for: maintaining, manipulatingand amplifying

differentDNA fragments.(pp 342 Griffiths et al 10e).

In the mid 1970

s the first methods for making recombinant DNA

molecules and transferring them toE. coliwere developed.

Currently, genes from practically every organism can be cloned,

identified, characterized and expressed. DNA can be even be cloned

from long dead organisms (100,000 years !) if the DNA is still

preserved.

Modifiedgenes can be reinserted into the genome of many species

the list of species for which this is possible is somewhat limited, but

growing.

3In 2010, a completely synthetic genome was inserted into a

Mycoplasma cell, a small prokaryote.


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Gene cloningrefers to theinsertion of foreign DNAinto a

vector,a molecular containerforaforeignfragmentof DNA,

achromosomethat can be replicated or cloned in a host cell.

Sequence:

1) recombinant DNA-a foreign fragment of DNA(gene ?) is insertedinto a vector.

2) The vector transports the gene into a host cell (bacteria?).

3) Within a host the vector may replicate and express the gene.

4) If the host cell divides, recombinant DNA is inherited.

5)If it survives, a colony or clone of identical host cells is produced

each with one or more recombinant vector chromosomes, or a

recombinant clone .

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(1)Cuttingachromosome or agenome into small

pieces.

(2) Insertingthechromosomefragment(s) intovectors.

(3)Getting the vectorswith fragments intobacteriahostsand in alibraryfor storage andreference.

(4) Identifying,

Indexingand selectingfrom a library.

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Forward analysis in

molecular genetics

(phenotype to genotype)

transformation

Marker

selection

cutting

inserting


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Foreign DNAforfragmentation and cloningmay be

obtained from:

Genomic DNAfrom the organism of interest, for

example, a Drosophila strain.

Complementary DNA- a double stranded,

complementary version of an mRNA molecule.It is

generated by reverse-transcribing DNAfrom an mRNA

template (using reverse-transcriptase).

Synthetic, or chemically- constructed DNA-

(Oligonucleotides)

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Endonucleases break internal phosphodiester bondswithin DNAstrands, many of them make random cuts.

The most useful restriction enzymesare (restriction)endonucleases which are site specific.Type II restriction

endonucleasescut DNA in aprecise way, only at specificnucleotide sequencescalled restriction or cut sites.

Restriction enzyme

is name that refers to their function,bacteria have these enzymes to restrict infectionbyforeign DNA by cutting and digesting it.

Bacteria protect their own DNA by methylation, recognized by itsown restriction enzymes

Cutting DNA in manageable pieces7


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Restriction Enzyme Nomenclature:first letter of the genus

(Escherichia), two letters of the species (coli), specific strain first

letter (Ry13) rank order of discovery (1) =EcoR1 8


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5GAATTC3

3

CTTAAG5

Restriction sites often have specific cuttingsequences withtwofold rotational symmetry. While this is not an actual

palindrome which has symmetry within a line, it is called aDNA palindrome

5GAATT

C3

3CTTAA

G5

5GAATTC3

3CTTAAG5

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Operator site 2 foldrotational symmetry

PREVIOUSLY ENCOUNTERED 2-FOLD

ROTATIONAL SYMMETRY

Inverted terminal repeatsequences identify family

in cut and pastetransposons

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The EcoRI enzyme will cut the recognition sequence

anywhere it encounters it in DNA.

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GATTAGCCGGAATTCTATACCGAC 3

3

CTAATCGGCCTTAAGATATGGCTG5

5

GATTAGCCGG 5

AATTCTATACCGAC 3

3

CTAATCGGCCTTAA5

GATATGGCTG 5

The short regions of single stranded DNA from cut

DNA are complementary,they overlap and they have the samelinkage site(3, 5).The overlapping ends are called

sticky

ends

.

They can re-anneal to complementary sequences to form double-

stranded DNA (with DNA ligase).

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(2) Pst I leaves 3' overhang - sticky ends joined by DNA ligase.5------------------------CTGCA G------------------3------------------------G ACGTC------------------

(3) Sma I leaves blunt endsjoined by a viral T4 DNA ligase.

-------CCC 3 GGG------

-------GGG 3CCC------To make a blunt end cut more useful

Add linkers(small DNA fragment with a restriction sequence) with T4 DNA

ligaseso the foreign DNA can be cut by a staggered endonuclease of yourchoice. CCCTGCA.. GGGGGG ACGTCCC

Thus a restriction site can be cut at or nearalmost any locationand specificrestriction target sequences engineered into a vector

(1) Eco RI digestion leaves 5 single stranded overhang.

5CTGCTAACG 3 AATTCCATCGTACT

3GACGATTGCTTAA 5

GGTAGCATGA

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Digestion of DNA -Predicting Fragment Sizes

Recognition sequences can be 4, 5, 6, and 8 base pair

long 6 base sites are those most often used.

The expected frequencyof a specific restriction site inany genomic DNA or cDNA is

(1/4)n

where n is the number of bases in the recognition sequence,that is, the number of cuts declines or fragments are larger onaverage with a longer base cutter.

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Expected frequencyof restriction sites in any long

piece of DNA:

Hae I GGCC ! x ! x !x ! = 1/256 bases

Tfi I GAATC (1/4) 5 = 1/1024 bases

Not I GCGGCCGC (1/4) 8"1/66,000

Bam HI GGATCC (1/4) 6 = 1/4096

TheAVERAGEobserved size of DNA digested by Bam

HI is about 4000 bp.

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What would be the average expected number of

bases DNA digested by Hind I which recognizes

the sequence:

GT(A or G) (T or C) AC ?

1/4 x 1/4 x 1/2 x 1/2 x 1/4 x 1/4 = 1/1024

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Assuming :(1) equal frequency of bases (GC = 50%),and (2) a random sequence of bases


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DNA is digested with restriction enzymes.

The size of a fragment affects its migration rate on a

polyacrylamide gel.

How do you measure the fragment size ?

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The size of DNA is readily measured by electrophoresis.

Put size markers in one well, cut genomic DNA with one or more

restriction enzymes, load it into the other wells and put it into an

electric field. Chromosome fragments move because they are

charged and carried with the polar buffer as it flows.

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Visualize (1) Radioactive label, (2) stain with EtBr, or

(3) use non mutagenic stains in the buffer of gel


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Agarose gel from

electrophoresisof DNA fragments

stained with

Ethidium bromide -

a powerfulmutagen.

Use non-mutagenic

dyes that can beincluded in the buffer

solution

8

4

2

1

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Electrophoresis

of DNA in anagarose gel

+

-1210

8

4

2

1

DNA fragments

migrate a distance

that is proportionalto the log of their molecular

weight (MW) in kilobasesafter

any fixed period of time.

D = (a-b) *(log M)M = molecular mass,

D= distance,

a &b = electrophoresis constants

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12

10

8

4

2

1

Molecular

Weight

Standards

(in kilo bases)

un- Eco Bgl Eco+

cut R1 Bgl

DNA Electrophoresis

Restriction site order

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7. 5

4. 5

2. 5

2. 0

Undigested: 12 kb

Eco RI: 2, 10kb

Bgl I : 4.5, 7.5kb

Eco RI + Bgl I :

2.0, 2.5, 7.5 kb

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(1)Eco RI gives 2 and 10 kb DNA

(2)Bgl I gives 4.5 and 7.5 kb DNA

(3) Bgl and Eco RI gives 2.0 , 2.5 kb and 7.5 kb.

2

The position of the sites are important which is in the middle?

Eco RI

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Bgl I

10

7.5L4.5L

120

or ?

4.5R7.5R

Restriction Map


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DNA of interest can be put into vectors,and cloned.

VECTORSare any DNA vehicle used for DNA replication

in an organism.DNA vectors differ in the fragment size they can carry including:

Smaller fragments

plasmids(5-15kb) circular

lambda bacteriophagecloning vectors (12-30kb) linearpackagedcosmid hybrid phage & plasmid (35-52kb) linearpackaged

Larger fragments- cloning vectors for eukaryotes

PACsor P1 artificial chromosome (80-100 kb)packaged, circular

BACsor bacterial (F) artificial chromosomes(150-300kb)circularbased on the F plasmid

YACsor yeast artificial chromosomes (300kb+)linear

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Plasmids in general

Most animals do not have circular plasmids, yeast andfew plants have them. Rare among other eukaryotes

Several of the following kinds of plasmids may be foundin one bacterial cell but they must be compatible- if not,

one will be lost from a cell.

F plasmids

R plasmids

Col plasmids- kill other bacteria

Degradative plasmids- degrade unusual chemicals orproteinVirulence Plasmids - confer pathogenecity

Ti Plasmid

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InsertCut:

(1) foreign DNA (with

Eco R1)

(2) cut the plasmid (with

Eco R1)

BindEnhance complementary

binding, DNA

polymerization, add

DNA ligase

Create:

a plasmid with foreignDNA (chimera) or

recombinant DNA

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staggered cutting a

specific sequence

means they can beprecisely excised and

and precisely ligated

into a plasmid - with

the same restriction

site.


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Once you have DNA in a vector, how do you

deliver the vector into Bacteria25


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A useful vector has (2) more than one insertion site

(3) Selection sites

(4) an origin of replication (ori)

and termination sequences

A useful vector is1: right size


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(1) the vector

contains multipleinsertion sites -

inside screening

or selection sites.

(2) cells containing

the vector can be

selected easily-

identified as

bacteria with arecombinant

plasmid

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Transformed bacterialcolonies in a mediumcontaining X gal.

When intact,galactosidase cleavesXgal and bacteria turnblue

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How amplification works, or why you need a replication origin

Figure 20-4

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Replicate

plasmids,

clone your

DNA if youhave a

replication

origin


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A useful vector :

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

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

fragmentthat can be cut by different endonucleases (restriction

sites)

(3) The vector has to be easily insertedin a host cell

(4) there are engineered selectable phenotypic characters, characters

that can be used to identify and select plasmids that actually

incorporated foreign DNA, usually disrupting a marker gene (a

selection site).

(5) an origin of replicationin the vector -so it can replicate

(6)Is it the right DNA ?You have a screening test for the gene of

interest.

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You screen for transformed bacteria, by selecting for

bacteria with fragment insertion markers.

How do you screenthe wide variety of fragments for a gene

or sequence of interest ?

(1) looking for an extrachromosomal foreign DNAsequence that can restore a mutant (mutant rescue) to awild-type phenotype (functional complementation)

or(2) molecular probes using complementary DNA or RNA

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TGTCTTTCCACTTCTCTTGGCTTGCTTTTGGAGGACCAGGTTGAG

Target DNA

ACAGAAAGTTGAAGAGAACCCAACGAAAACCTCTTCTTCCAACTC

DNA hybridization (and ID) by complementary binding.

Two single stranded pieces of DNA that are

complementary will spontaneously form a double helix

ACAGAAAGTTGAAGAGAACCCAACGAAAACCTCTTCTTCCAACTC

32P 32P 32P

32P 32P 32P

TGTCTTTCACCTTCTCTTGGCTTGCTTTTGGAGGACCAGGTTGAG

||||||||| |||||||||| |||||||||||| || ||||||||

Even if the match is not perfect.

Probe DNA

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Getting asequence ofinterest froma phagelibrarybyfinding theclone ofinterest byusing DNA orRNA probesthathybridize

with specific(target)plaquesorcolonies

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Lyse phage and

denature DNA

(NaOH)

or a

florescent

tag


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Where do you get the DNA or RNA probes ?

(1) A homologous genefrom a related organism.

(2) mRNA synthesizedfrom a minimally degenerate

amino acid sequence of a known protein.

(3) mRNA collected from a tissueexpressing a specific

protein.

(4) Complementary DNA(cDNA) isolated from a tissue

with a high level of a specific gene expression, made

with reverse transcriptase(5) Another clone

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A useful vector :

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

(2) there are (engineered) restriction sitesin the vector and thefragmentthat can be cut by different endonucleases (restriction

sites)

(3) an origin of replicationin the vector -so it can replicate

(4) there are engineered selectable phenotypic characters, charactersthat can be used to identify and select plasmids that actually

incorporated foreign DNA, usually disrupting a marker gene (a

selection site).

(5) The vector has to be easily insertedin a host cell

(6) You have a screening testfor the gene of interest.

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If the goal is not just cloning a fragment or identifying a gene, but

expressing a protein, a foreign gene is inserted into an expression

vector containing transcription & translation sequences ( bacterial

promoter, initiation and termination sequences regulator andoperator genes, ribosome binding site, etc. )

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L17Biol261FragInsertDNA2013