What are the Techniques of Biotechnology ?

• Restriction Endonucleases: enzymes that cut DNA at

specific codes (nucleotide sequences)– Can buy from suppliers: ex. cut at ATATAT

• DNA Fingerprinting: sequence code of sample DNA (a portion of genome) or compare digested samples via gel electrophoresis

– In crime and paternity testing, evidence sample compared to suspects’ samples; if different codes, or different patterns on gel, then cannot be “donor”; if match, % likelihood based on size of genome sequenced, or frequency of gel pattern in population

• Rape charges even filed against “unknown person” with sample DNA (statute of limitations was approaching)

• DNA very strong evidence for innocence, not as strong for guilt; but has been used as primary evidence in capital cases (resulted in executions)

– DNA from whale-meat in Japanese restaurants showed many whale and dolphin species sold despite moratorium on most

species• Polymerase chain reaction: machine that replicates a small

sample of DNA into a larger amount of identical sample (enough to work with)

Fig. 20.3Restriction site

DNA

Sticky end

Restriction enzymecuts sugar-phosphatebackbones.

53

35

1

One possible combination

Recombinant DNA molecule

DNA ligaseseals strands.

3

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

2

Fig. 20.2

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Plasmid

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

Gene for pest resistance inserted into plants

Gene used to alter bacteria for cleaning up toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

2

4

1

3

Fig. 20.6DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

DNA polymerase

cDNA

Fig. 20.9aMixture ofDNA mol-ecules ofdifferentsizes

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

1

2

Powersource

– +

+–

Fig. 20.8

5

Genomic DNA

TECHNIQUE

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Cycle 2yields

4molecules

Cycle 3yields 8

molecules;2 molecules

(in whiteboxes)

match targetsequence

Targetsequence

Primers

Newnucleo-tides

3

3

3

3

5

5

51

2

3

What are the Applications of

Biotechnology? • Human Genome Project: highly collaborative; completed in early 2001;

human genome mapped and sequenced; next step is understanding the functions of genes (and resulting proteins)– Reasoning was that several genetic diseases would become better under-

stood during the project (sooner than if each was studied independently)

• Genetic Screening: geneticists use interviews and DNA fingerprinting; concerns regarding insurance and potential discrimination

• Genetic Therapy: inject “healthy genes” into blood; some success in diseases of the blood (immune disorders)

• Genetic Engineering (recombinant technology): manipulate genes in fertilized egg; replace un-wanted gene with copy of desired gene – Transgenic Organisms: because genetic code and ribosome “machinery”

shared in all organisms, bacteria (and other organisms) can make human proteins if appropriate gene is inserted into cell (or fertilized egg); such proteins are often medicines (ex., replace casein gene in milk of sheep or goats with desired gene)

– Agricultural Applications: genes for natural insecticides, drought-resistance, and frost-resistance transferred to crops

Fig. 21.2

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Linkage mapping1

2

3

Geneticmarkers

Physical mapping

Overlappingfragments

DNA sequencing

Fig. 21.3

Cut the DNAinto overlappingfragments short enoughfor sequencing

1

2

3

4

Clone the fragmentsin plasmid or phagevectors.

Sequence eachfragment.

Order thesequences intoone overallsequencewith computer software.

Fig. 20.10

Normalallele

Sickle-cellallele

Largefragment

(b) Electrophoresis of restriction fragments from normal and sickle-cell alleles

201 bp175 bp

376 bp

(a) DdeI restriction sites in normal and sickle-cell alleles of -globin gene

Normal -globin allele

Sickle-cell mutant -globin allele

DdeI

Large fragment

Large fragment

376 bp

201 bp175 bp

DdeIDdeI

DdeI DdeI DdeI DdeI

Fig. 20.22Bone

marrow

Clonedgene

Bonemarrowcell frompatient

Insert RNA version of normal alleleinto retrovirus.

Retroviruscapsid

Viral RNA

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.

1

2

3

4

Fig. 20.25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS