Figure 20.2 Overview of gene cloning with a bacterial plasmid, showing various uses of cloned genes

Bacterium

Bacterialchromosome

Plasmid

Cell containing geneof interestGene inserted

into plasmid

RecombinantDNA (plasmid) Plasmid put into

bacterial cell

Gene of interest

DNA ofchromosome

Recombinatebacterium

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

Protein harvested

Basic research on protein

Basic research and various applications

Gene of interest

Copies of gene

Basic research on gene

Gene for pestresistance inserted into plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hormone treatsstunted growth

Protein expressedby gene of interest

1

2

3

4

Figure 20.3 Using a restriction enzyme and DNA ligase to make recombinant DNA

Restriction site

DNA 53 5

3G A A T T CC T T A A G

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow

DNA fragment from another source is added. Base pairing of sticky ends produces various combinations.

DNA ligaseseals the strands.

Sticky end

Fragment from differentDNA molecule cut by thesame restriction enzyme

One possible combination

Recombinant DNA molecule

G

C T T A AA A T T C

G

A A T T C

C T T A AG

G

G GA A T T C A A T T C

C T T A A G C T T A A G

1

2

3

Figure 12.3 Cloning a gene in a bacterial plasmid E.coli

Plasmid

Isolate DNAfrom two sources

Cut both DNAswith the samerestriction enzyme

Human cell

DNA

2

1

3

4

5

6

Gene V

Sticky ends

Mix the DNAs;they join bybase-pairing

Add DNA ligaseto bond the DNA covalently

Recombinant DNAplasmid Gene V

Put plasmid into bacteriumby transformation

Recombinant bacterium

Clone the bacterium

Bacterial clone carrying manycopies of the human gene

Genomic libraries

Recombinantplasmid

Genome cut up withrestriction enzyme

Recombinantphage DNA

or

Bacterialclone

Phageclone

Phage libraryPlasmid library

Making an intron-lacking gene from eukaryotic mRNA

Cell nucleus

DNA ofeukaryoticgene

Exon Intron Exon Intron Exon

1

2

3

4

5

Transcription

RNA splicing(removes introns)

Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

Breakdown of RNA

Synthesis of secondDNA strand

RNA transcript

mRNA

Reverse transcriptase

cDNA strand

cDNA of gene(no introns)

Test tube

Table 12.6 Some protein products of recombinant DNA technology

A DNA probe tags a gene by base pairing

Radioactiveprobe (DNA)

Single-strandedDNA

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Base pairingindicates thegene of interest

A T C C G A

A T G C G C T T A T C G

A G C

C T

T A

T G

C A

T

A T C C G A

A G G

T A G G

C T A A

PCRTargetsequence

53

5

Genomic DNA

Denaturation:Heat brieflyto separate DNA strands

Annealing: Cool to allow primers to hydrogen-bond.

Extension:DNA polymeraseadds nucleotidesto the 3 end of each primer

Cycle 1yields

2 molecules

Cycle 2yields

4 molecules

Cycle 3yields 8

molecules;2 molecules

(in white boxes)match target

sequence

5

3

3

5

Primers

Newnucleo-tides

1

2

3

3

PCR

Primitive PCR machine

http://www.edvotek.com/images/532.jpg

Applications for PCR

• DNA cloning for sequencing

• Functional analysis of genes

• Diagnosis of genetic diseases

• ID genetic fingerprints (i.e. forensics and paternity testing)

• Detection and diagnosis of infectious diseases (e.g. H1N1)

Gel electrophoresis of DNA

+ +

– –

Powersource

Gel

Mixture of DNAmolecules ofdifferent sizes

Longermolecules

Shortermolecules

Completed gel

Lane 1 – Father

Lane 2 – Child

Lane 3 – Mother

The child has inherited some, but not all of the fingerprint of each of its parents, giving it a new and unique fingerprint.

Gel box and power source

Gel Electrophoresis Plate

DNA fingerprints from a murder caseDefendant’sblood (D)

Blood fromdefendant’sclothes

Victim’sblood (V)

D Jeans shirt V

4 g 8 g

“Pharm” animals