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
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