Chapter 14: Genetic Engineering -Modification of the DNA of an organism to produce new genes with...

Chapter 14: Genetic Engineering

-Modification of the DNA of an organism to produce new genes with new

characteristics

Biotechnology

• Use of organisms to benefit humanity

Recombinant DNA technology

• DNA from different organisms is spliced together

• Allows scientists to make many copies of any DNA segment (clone)

• Can introduce foreign DNA into cells of microorganisms

Recombinant DNA technology• Restriction enzymes – cut DNA– Bacteria produce for defense against viruses

• Vector – transports DNA into a cell– Ex: bacteriophage– Plasmid – separate, smaller circular DNA that

maybe be present and able to replicate inside bacteria

• Transformation – uptake of foreign DNA by cells– How plasmids can get into bacteria

Bacterial Conjugation

and Recombination

Recombinant DNA technology• Palindromic sequences – reads the same as

complement, in opposite direction– AAGCTT– TTCGAA– Many restriction enzymes cut these sequences

• Restriction enzymes cut on a stagger sticky ends (can pair with complementary single-stranded end of other DNA cut with same enzyme)

• DNA Ligase – links 2 fragments recombinant DNA

Fig. 20-3-3Restriction 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

Restriction Enzymes

Steps of Creating a Recombinant DNA Plasmid (Basic)

• 1. Plasmids and desired DNA cut by same restriction enzyme

• 2. Mix 2 types of DNA so sticky ends pair• 3. DNA ligase forms bonds between fragments

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

Cloning a Gene

Fig. 20-4-4

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Bacteria carryingplasmids

RESULTS

Colony carrying non-recombinant plasmidwith intact lacZ gene

One of manybacterialclones

Colony carrying recombinant plasmid with disrupted lacZ gene

Cloning DNA

• Genome – total DNA per cell• Genomic library – collection of DNA fragments

more or less representative of all DNA in genome

• Genetic Probe – single stranded DNA or RNA that is radioactively labeled and can attach to target sequence by base pairing rules

• A probe can be synthesized that is complementary to the gene of interest

• For example, if the desired gene is

– Then we would synthesize this probe G5 3… …G GC C CT T TA A A

C3 5C CG G GA A AT T T

DNA Probe

Using a DNA probe

Polymerase Chain Reaction (PCR)

• Can amplify a small sample of DNA quickly• DNA replication in vitro• 2 strands separated by heating so special

heat-resistant DNA polymerase called Taq polymerase used (thermophile)

• MAJOR BONUS: Only specific sequences can be replicated

• Study: crime scenes, archaeological remains

PCR

Gel Electrophoresis• Separates fragments like DNA, RNA or

polypeptides (they carry charge and can migrate in an electrical field

• RNA and DNA (-) --- so they move to (+) pole• Smaller fragments go further• Compare sample to standard• Usually “blot” - transfer DNA from gel to

nitrocellulose filter for further analysis• DNA Fingerprinting

Gel Electrophoresis

Fig. 20-9a

Mixture ofDNA mol-ecules ofdifferentsizes

Powersource

Longermolecules

Shortermolecules

Gel

AnodeCathode

TECHNIQUE

1

2

Powersource

– +

+–

Fig. 20-9b

RESULTS

DNA Fingerprint

Transgenic Organisms

• Plants and animals in which foreign genes have been incorporated

• Animals– Inject DNA into nucleus of egg or stem cell– Eggs implanted in uterus; stem cells injected into

blastocysts + then implanted into foster mother

• Plants– Disease resistance– Pesticide resistance

Transgenics

Fig. 20-20

Culturedstem cells

Early human embryoat blastocyst stage

(mammalian equiva-lent of blastula)

Differentcultureconditions

Differenttypes ofdifferentiatedcells

Blood cellsNerve cellsLiver cells

Cells generatingall embryoniccell types

Adult stem cells

Cells generatingsome cell types

Embryonic stem cells

From bone marrowin this example

Fig. 20-18

TECHNIQUE

Mammarycell donor

RESULTS

Surrogatemother

Nucleus frommammary cell

Culturedmammary cells

Implantedin uterusof a thirdsheep

Early embryo

Nucleusremoved

Egg celldonor

Embryonicdevelopment Lamb (“Dolly”)

genetically identical tomammary cell donor

Egg cellfrom ovary

Cells fused

Grown inculture

1

33

4

5

6

2

Fig. 20-19

Cloning Video

GE Plants

Application of GE

• Human proteins– Insulin– Hormones - HGH

• Human treatments for disease– Multiple sclerosis, certain cancers, heart attacks,

forms of anemia

• Vaccines

Fig. 20-23

Fig. 20-24This photo shows EarlWashington just before his release in 2001,after 17 years in prison.

These and other STR data exonerated Washington andled Tinsley to plead guilty to the murder.

(a)

Semen on victim

Earl Washington

Source of sample

Kenneth Tinsley

STRmarker 1

STRmarker 2

STRmarker 3

(b)

17, 19

16, 18

17, 19

13, 16 12, 12

14, 15 11, 12

13, 16 12, 12

Forensics