Chapter 20:Biotechnology

2. DNA Cloning

1. DNA Sequencing

3. Studying Gene Expression

4. Manipulating Genomes

5. Therapeutic & DiagnosticTechniques

1. DNA SequencingChapter Reading – pp. 409-412

DNA(template strand)

TECHNIQUEPrimer Deoxyribonucleotides Dideoxyribonucleotides

(fluorescently tagged)

DNApolymerase

5′

5′

3′

3′

OH H

GG

dATP

dCTP

dTTP

dGTP

P P P P P P

ddATPddCTP

ddTTP

ddGTP

T

TT

G

G

G

C

C

C

CT

TT

A

A

AA

DNA Sequencing…

• DNA replication in vitro using 1 of 4 different “chain-terminating” dideoxynucleotides (ddNTPs)

• results in a set of DNA fragments ending in all positions with A, C, G, or T that can be resolve by length on a gel

DNA Sequencing uses Chain Terminators

Normal nucleotide (dNTP)

Dideoxynucleotide (ddNTP)

*

DNA synthesis is carried out in reactions containing the following:

• DNA template to be sequenced

• dNTP’s

• DNA primer

• DNA polymerase

• ddATP, ddCTP, ddGTP or ddTTP

• each ddNTP is labeled with a different fluorescent tag

The color reveals the identity of the ddNTP that terminated DNA synthesis at each position,

thus revealing the sequence!

DNA (templatestrand)

Labeled strands

Shortest LongestDirectionof movementof strands

Longest labeled strand

Detector

LaserShortest labeled strand

TECHNIQUE (continued)

5′

3′

G

G

C

C

C

CT

TT

A

A

AA

T

TT

GddC

ddC

ddG

ddG

ddG

ddA ddA

ddAddT

3′

5′

T

TT

G

CT

TT

GCG

TTT

GCGA

TTT

GCGAA

TTTG

CGAAG

TTT

CGAAGT

TTT

CGAAGTC

A

T

TT

CGAAGTC

G G G

…DNA Sequencing…

RESULTSLast nucleotideof longestlabeled strand

Last nucleotideof shortestlabeled strand

G

G

G

A

AA

C

C

T

Directionof movementof strands

Longest labeled strand

Detector

LaserShortest labeled strand

…DNA Sequencing

“Next Generation” Sequencing

2. DNA CloningChapter Reading – pp. 409-417

What is “Recombinant DNA”?The joining of DNA from different sources.

This can happen in nature (in vivo)…• the transfer of DNA involving bacteria or viruses

…or in the laboratory (in vitro)• the cutting & splicing of DNA fragments by

molecular biologists

The term “recombinant DNA” generally refers to the in vitro kind which is commonly called “gene cloning”…

Bacterium

Bacterialchromosome

Plasmid

2

1 Gene inserted intoplasmid Cell containing gene of interest

RecombinantDNA (plasmid)

Gene of interest

Plasmid put intobacterial cell

DNA ofchromosome(“foreign” DNA)

Recombinantbacterium

Gene Cloning…

• gene of interest is inserted into a plasmid vector• bacterial host transformed with recombinant plasmid• bacterial clone with recombinant plasmid is identified

Host cell grown in culture to form a clone of cells containing the “cloned” gene of interest

Gene of interest

Protein expressed fromgene of interest

Protein harvestedCopies of gene

Basic researchand variousapplications

3

4Basicresearchon protein

Basic research on gene

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growthhormone treatsstunted growth

…Gene CloningCloned gene can then be used in a variety of ways.

Recombinant DNA molecule

One possible combinationDNA ligaseseals strands

DNA fragment addedfrom another moleculecut by same enzyme.Base pairing occurs.

Restriction enzymecuts sugar-phosphatebackbones.

Restriction site

DNA5′

5′

5′

5′

5′

5′

5′

5′

5′5′

5′

5′

5′5′

5′

5′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

3′

2

3

1

Sticky end

GAATTCCTTAAG

GG

GG

AATT CAATT CC TTAA C TTAA

Gene cloning usually involves the use of restriction enzymes that cut DNA at very specific sequences:

Restriction Enzymes

e.g. EcoRI cuts at:..GAATTC....CTTAAG..

There are many different restriction enzymes,

each cutting a different sequence

Bacterial plasmidTECHNIQUE

ampR gene lacZ gene

Restrictionsite

Hummingbird cell

Sticky ends Gene ofinterest

Humming-bird DNAfragments

Recombinant plasmids Nonrecombinant plasmid

Bacteria carryingplasmids

The Gene Cloning Technique

• cut plasmid & DNA to be cloned with same RE

• ligate vector &DNA insert withDNA ligase

• transform bacteria with ligated DNA

RESULTS

Bacteria carryingplasmids

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carrying recombinantplasmidwith disruptedlacZ gene

One of manybacterialclones

Selection for Bacterial ClonesAntibiotic resistance allows selection for bacterial clones containing the plasmid vector.

Blue/white selection via lacZ gene disruption allows identification of clones containing a DNA insert.

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Powersource

Longermolecules

Cathode Anode

Wells

Gel

Shortermolecules

TECHNIQUE

RESULTS

1

2

− +

− +

Gel ElectrophoresisSeparation of DNA fragments through a porous gel matrix:

• gel is either agarose orpolyacrylamide

• electric current pulls negatively charged DNA toward the positive pole

• rate of movement is inversely proportional to DNA fragment size

DNA Libraries…A collection of cloned genes from an organism is called a DNA library.

Genomic DNA Library• a collection of chromosomal DNA fragments clonedinto a particular vector

cDNA Library• a collection of DNA fragments produced from messenger RNA (mRNA) cloned into a vector

• essentially cloned pieces of the organism’s genome

• produced from mRNA by reverse transcriptase• a collection of expressed genes with NO intron DNA

Foreign genome

Cut with restriction enzymes into eithersmallfragments

largefragments

or

Recombinantplasmids

Plasmidclone

(a) Plasmid library

(b) BAC clone

Bacterial artificialchromosome (BAC)

Largeinsertwithmanygenes

(c) Storing genome libraries

…DNA Libraries

DNA innucleus

mRNAs incytoplasm

mRNAReversetranscriptase Poly-A tail

DNAstrand

Primer

DNA polymerase

cDNA

5′5′

5′5′

5′5′

5′5′

3′3′

3′3′

3′3′

3′3′

A A A A A A

A A A A A A

T T T T T

T T T T T

Producing cDNAcDNA (DNA complementary to mRNA) is produced as follows:

• purify mRNA fr. desired cell type• treat with reverse transcriptaseand oligo-dT primer

• results in double-stranded DNA copies of all mRNA from the cell

• clone cDNA fragments into vector such as a plasmid

Results in a collection of cloned cDNA corresponding to coding

sequences of all expressed proteins

• transform bacterial hosts

Radioactivelylabeled probemolecules Gene of

interestProbeDNA

Single-strandedDNA fromcell

Film

Location ofDNA with thecomplementarysequence

Nylonmembrane

Nylon membrane

Multiwell platesholding libraryclones

TECHNIQUE 5′

5′3′

3′

GAGTAGTGGCCG⋅⋅⋅CTCATCACCGGC⋅⋅⋅

Screening DNA Libraries

• DNA from library clones immobilized on a membrane• radioactively labeled DNA similar in sequence to desired clone is added as a probe

• hybridization of probe to complementary DNA IDs clone

Genomic DNA

Targetsequence

Denaturation

Annealing

Extension

Primers

Newnucleotides

Cycle 1yields

2molecules

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

5′

5′

5′

5′

3′

3′

3′

3′

2

3

1

TECHNIQUEPCRThe Polymerase Chain Reaction is a technique to selectively amplify a desired DNA sequence:

• essentially DNA replicationin vitro

• results in exponentialamplification of target DNA sequence

• artificial primers specificfor target DNA sequencelimit replication to DNAcomplementary to primers

Overview of PCR

2) artificial primers “flanking” DNA of interest

3) heat-stable DNA polymerase (from hyperthermophile)

Every PCR reaction requires the following:1) source of target DNA template

4) dNTP’s

5) automated thermocycler to facilitate repeated:• denaturation of DNA (separating the 2 strands)• hybridization of primers to template• DNA synthesis

Resulting PCR products can then be analyzed by gel electrophoresis.

“PCR Cloning” involves designing restriction

sites into the primers to facilitate cloning

3. Studying Gene ExpressionChapter Reading – pp. 417-419, 421-422

cDNA synthesis

PCR amplification

Gel electrophoresis

mRNAs

cDNAsPrimers

β-globingene

Embryonic stages1 2 3 4 5 6

2

3

1

RESULTS

TECHNIQUE RT-PCR• produce cDNAwith reverse transcriptase

• carry out PCR using specific primers for desired gene

• gel electrophoresisof resulting PCR fragments

Amount of DNA reflects amount of

original mRNA sequence.

in situ Hybridization

…in situ Hybridization

• fluorescently labeled anti-sense RNA probes added to tissue sample

• hybridization with complementary mRNA sequences

• location of specific gene expression within the tissue (i.e., in situ) is revealed

How is in situ hybridization carried out?

DNA MicroarraysA DNA microarray is a solid surface containing a precise array of single-stranded DNA sequences from 1000s of different genes in an organism.

• labeled cDNA is produced from test cells and allowed to hybridize with sequences in the array

• intensity of signals reveal expression of specific genes within the test cells.

When cDNA from different sources is labeled differently, gene expression from each source can be compared in a

single microarray (as shown on the slide after next).

Isolate mRNA.

2

1

3

4

TECHNIQUE

Make cDNA by reversetranscription, usingfluorescently labelednucleotides.

Apply the cDNA mixture to a microarray, a different genein each spot. The cDNA hybridizeswith any complementary DNA onthe microarray.

Rinse off excess cDNA; scan microarrayfor fluorescence. Each fluorescent spot(yellow) represents a gene expressedin the tissue sample.

Tissue sample

mRNA molecules

Labeled cDNA molecules(single strands)

DNA fragmentsrepresenting aspecific gene

DNA microarray

DNA microarraywith 2,400human genes

Analyzing gene

expression using a DNA microarray.

Comparing Gene Expression

DNA

SNPNormal allele

Disease-causingallele

T

C

Single Nucleotide Polymorphisms (SNPs)

SNPs are single nucleotide differences that correspond with specific disease-causing alleles.

PCR and hybridization techniques (e.g., microarrays) can reveal the presence of such alleles (genetic testing).

4. Manipulating GenomesChapter Reading – pp. 422-425, 429-430

Crosssection ofcarrot root

2-mgfragments

Fragments werecultured in nu-trient medium;stirring causedsingle cells toshear off intothe liquid.

Single cellsfree insuspensionbegan todivide.

Embryonicplant developedfrom a culturedsingle cell.

Plantlet wascultured onagar medium.Later it wasplanted in soil.

Adultplant

Cloning PlantsSome plants can be cloned from a single adult plant cell.

Frog embryo Frog egg cell Frog tadpole

UV

Less differ-entiated cell

Donornucleustrans-planted

Enucleatedegg cell

Fully differ-entiated(intestinal) cell

Donornucleustrans-plantedEgg with donor nucleus

activated to begindevelopment

Most developinto tadpoles.

Most stop developingbefore tadpole stage.

EXPERIMENT

RESULTS

This experiment produced the first

artificially cloned animal.

Cloning Animals

Mammarycell donor

21

3

4

5

6

TECHNIQUE

RESULTS

Culturedmammarycells

Eggcell fromovary

Egg cell donor

NucleusremovedCells fused

Grown in culture

Implanted in uterusof a third sheep

Embryonicdevelopment

Nucleus frommammary cell

Early embryo

Surrogatemother

Lamb (“Dolly”) geneticallyidentical to mammary cell donor

The first cloned mammal, “Dolly the sheep”, was cloned by this method.

Other mammals such as cats have since been cloned and presumably humans could be cloned this way as well.

Transgenic OrganismsTransgenic organisms have a foreign gene (e.g. from another species) inserted into their genome.

• also known as “genetically modified organisms” (GMOs)

Cas9 active sitesGuide RNAcomplementarysequence

Cas9 protein Guide RNA engineered to“guide” the Cas9 proteinto a target gene

5′

5′3′

5′

NUCLEUS

3′5′

3′

Part of thetarget gene

Resulting cut in target gene by

Cas9Active sites thatcan cut DNA

Cas9–guide RNA complex

Complementarysequence that canbind to a target gene

Normal(functional)gene for use asa template byrepair enzymes

CYTOPLASM

(a) Scientists can disable(“knock out”) the target geneto study its normal function.

(b) If the target gene hasa mutation, it can berepaired.

NUCLEUSRandom nucleotides Normal nucleotides

13

2

CRISPR-Cas9• a new method to modify genomes

5. Therapeutic & Diagnostic Techniques

Chapter Reading – pp. 425-431

Culturedstem cells

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Embryonicstem cells

Adultstem cells

Cells generatingall embryoniccell types

Cells generatingsome cell types

Livercells

Nervecells

Bloodcells

Stem CellsStem cells are undifferentiated cells that can give rise to multiple differentiated cell types.

Embryonic stem cells are considered more pluripotent than adult stem cells, though techniques are being developed to expand the potential of adult stem cell.

Remove skin cellsfrom patient. 2

1

3

4

Reprogram skin cellsso the cells becomeinduced pluripotentstem (iPS) cells.

Patient withdamaged hearttissue or otherdisease

Return cells topatient, wherethey can repairdamaged tissue.

Treat iPS cells sothat they differentiateinto a specificcell type.

Stem Cell Therapy

Techniques are being developed to treat a person’s own cells in vitro to produce a needed cell type which is then reintroduce into the patient to repair damaged tissue.

Cloned gene

2

1

3

4

Retroviruscapsid

Bonemarrowcell frompatient

Viral RNA

Bonemarrow

Insert RNA version of normal alleleinto retrovirus.

Let retrovirus infect bone marrow cellsthat have been removed from thepatient and cultured.

Viral DNA carrying the normalallele inserts into chromosome.

Inject engineeredcells into patient.

Gene Therapy

Bone marrow stem cells can be genetically modified in vitro to fix a genetic defect and then reintroduced back into the patient.

Normal β-globin allele

Sickle-cell mutant β-globin allele

Largefragment

Normalallele

Sickle-cellallele

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal andsickle-cell alleles of the β-globin gene

(b) Electrophoresis of restrictionfragments from normal andsickle-cell alleles

201 bp175 bp

376 bp

Large fragment

Large fragment

DdeI DdeI DdeI DdeI

DdeI DdeI DdeI

Restriction Fragment Length Polymorphisms (RFLPs)

• DNA from different sources cut with the same RE willresult in different size DNA fragments (RFLPs)

This photo showsWashington just beforehis release in 2001,after 17 years in prison.

(a)

(b) These and other STR data exonerated Washingtonand led Tinsley to plead guilty to the murder.

Semen on victim

Earl Washington

Kenneth Tinsley

17,19

16,18

17,19

13,16

14,15

13,16

12,12

11,12

12,12

Source ofsample

STRmarker 1

STRmarker 2

STRmarker 3

Short Tandem Repeat AnalysisMany parts of the human genome contain short DNA sequences repeated many times in a row (in tandem)

• the number of repeats in these regions is highly variable from person to person

• RFLP analysis can produce a DNAfingerprint that is unique for each individual

DNA + restriction enzyme

321

4

TECHNIQUE

I Normalβ-globinallele

II Sickle-cellallele

III Heterozygote

Restrictionfragments Nitrocellulosemembrane (blot)

Heavyweight

Gel

Sponge

Alkalinesolution Paper

towels

III III

III III III III

Preparation ofrestriction fragments

Gel electrophoresis DNA transfer (blotting)

Radioactively labeledprobe for β-globingene

Nitrocellulose blot

Probe base-pairswith fragments

Fragment from sickle-cellβ-globin allele

Fragment from normal β- globin allele

Filmoverblot

Hybridization with labeled probe Probe detection5

Southern Blotting

Key Terms for Chapter 20

• genomic & cDNA libraries, hybridization, probe

• recombinant DNA, gene cloning, plasmid vector• transformation, ligation, restriction enzymes

• gel electrophoresis, PCR, RT-PCR• dideoxynucleotide, RFLP, STR, SNP• in situ hybridization, DNA microarrays• transgenic, stem cell, southern blot, CRISPR-Cas9• in vivo, in vitro, in situ Relevant Chapter

Questions 1-10, 12

Slide Number 1Slide Number 2Slide Number 3Slide Number 4Slide Number 5Slide Number 6Slide Number 7Slide Number 8Slide Number 9Slide Number 10Slide Number 11Slide Number 12Slide Number 13Slide Number 14Slide Number 15Slide Number 16Slide Number 17Slide Number 18Slide Number 19Slide Number 20Slide Number 21Slide Number 22Slide Number 23Slide Number 24Slide Number 25Slide Number 26Slide Number 27Slide Number 28Slide Number 29Slide Number 30Slide Number 31Slide Number 32Slide Number 33Slide Number 34Slide Number 35Slide Number 36Slide Number 37Slide Number 38Slide Number 39Slide Number 40Slide Number 41Slide Number 42Slide Number 43Slide Number 44Slide Number 45