Biotechnology andGenetic Engineering

AP Biology Chapter 20

Terminology

• Genetic engineering – direct manipulation of genetic material for practical purposes

• Biotechnology – use of living organisms or their components to make products for us

• Recombinant DNA – combining pieces of DNA from different organisms

• Gene cloning – making copies of DNA

Making recombinant DNA

• Plasmids (small circular pieces of DNA in bacterial cells) are used to insert pieces of foreign DNA

The DNA is cut using restriction enzymes

What are restriction enzymes?

• Restriction enzymes come from bacteria and recognize a particular pattern of DNA, often 4, 6 or 8 base pairs long, and then cut the DNA within this recognized sequence.

• Bacteria use these enzymes to kill off other competing bacteria by cutting up their DNA.

How do they cut?

STICKY ENDS BLUNT ENDS

ACT GAA TTC CGG AAT GAA TTC

TGA CTT AAG GCC TTA CTT AAG

Where would the enzyme EcoRI cut?

ACT GAA TTC CGG AAT GAA TTC

TGA CTT AAG GCC TTA CTT AAG

There would be three pieces: one 4 bases, one 12 bases, and one 5 bases.

How do bacteria protect it’s own DNA from being cut by

the enzymes?

It methylates it’s own DNA.

Making recombinant DNA in plasmids

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/steps_in_cloning_a_gene.html

http://www.nearingzero.net/natural/screenres/natural039.jpg

Bacterial plasmids often contain antibiotic resistance genes.

Genes can be cloned into

vectors such as plasmids

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. 20-2a

DNA of chromosome

Cell containing geneof interest

Gene inserted intoplasmid

Plasmid put intobacterial cell

RecombinantDNA (plasmid)

Recombinantbacterium

Bacterialchromosome

Bacterium

Gene ofinterest

Plasmid

2

1

2

Fig. 20-2b

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

Gene ofInterest

Protein expressedby gene of interest

Basic research andvarious applications

Copies of gene Protein harvested

Basicresearchon gene

Basicresearchon protein

4

Recombinantbacterium

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

3

Fig. 20-4-1

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Fig. 20-4-2

Bacterial cell

Bacterial plasmid

lacZ gene

Hummingbird cell

Gene of interest

Hummingbird DNA fragments

Restrictionsite

Stickyends

ampR gene

TECHNIQUE

Recombinant plasmids

Nonrecombinant plasmid

Fig. 20-4-3

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

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

Steps

1. Plasmid and DNA of gene of interest are isolated.

2. Both DNAs are cut with the same restriction enzyme.

3. “new” DNA is ligated into plasmid4. Recombinant plasmids are inserted

into bacterial cells.5. Plate bacteria on agar. Bacteria will

express new genes.

Plasmid Maps

Plasmid MapsSometimes called restriction maps are graphical

representation of plasmids, that show the locations of major identifiable landmarks on DNA like restriction enzyme sites, genes of interest, plasmid length etc.

The collection of thousands of clones of bacteria containing recombinant plasmids is called a genomic library.

• In molecular biology, plasmid (or restriction) maps are used as a reference to engineer plasmids.

• The plasmids are digested by enzymes chosen and the resulting samples are subsequently run on an electrophoresis gel.

Our experiment: to transform E.coli with pGLO plasmid containing the jellyfish gene GFP to make them have the ability to glow

• To isolate only the cells containing the pGLO DNA, the plasmid contains the beta-lactamase gene which encodes for an ampicillin resistance (Ampr) protein.

• After the transformation, the cells are grown on a solid medium called an agar plate. This medium will contain the antibiotic ampicillin.

• In the presence of the ampicillin, only the bacteria containing the pGLO plasmid will have the Ampr protein which will break down the antibiotic, and be able to grow.

• This process is called antibiotic selection.

GFP results in E.coli

This plate shows bacteria expressing six different types of flourescent proteins

GRP has been used as tracers to see if the plasmid has been taken up by

the bacteria.

How much does it cost to make a transgenic mouse?

• Transgenic Mouse Production: The current fee for transgenic mouse production for UTMB

• investigators is $4000 (for 3 days of injections into [C57BL/6 X C3H/He]F2 embryos) or $5200

• (for 4 days of injection into C57BL/6 or FVB/N embryos; $1250 for each additional day

Storing Cloned Genes in DNA Libraries

• Plasmid libraries containing genes of interest cloned in

• Phage library that is made using bacteriophages which store genes of interest

Fig. 20-5a

Bacterial clones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

(a) Plasmid library (b) Phage library

Phageclones

Viruses used

as vectors

• BACs (bacterial artificial chromosome) are another type of vector used in DNA library construction

• A bacterial artificial chromosome (BAC) is a large plasmid that has been trimmed down and can carry a large DNA insert

Fig. 20-5

Bacterial clones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

(a) Plasmid library (b) Phage library (c) A library of bacterial artificial chromosome (BAC) clones

Phageclones

Large plasmidLarge insertwith many genes

BACclone

• A complementary DNA (cDNA) library is made by cloning DNA made in vitro by reverse transcription of all the mRNA produced by a particular cell

• A cDNA library represents only part of the genome—only the subset of genes transcribed into mRNA in the original cells

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/cdna.html

Fig. 20-6-1

DNA innucleus

mRNAs in cytoplasm

Fig. 20-6-2

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

Fig. 20-6-3

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

Fig. 20-6-4

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

DNA polymerase

Fig. 20-6-5

DNA innucleus

mRNAs in cytoplasm

Reversetranscriptase Poly-A tail

DNAstrand

Primer

mRNA

DegradedmRNA

DNA polymerase

cDNA

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/fish.html

Ways to introduce

new genes into bacteria.

Conjugation – through tubes between bacteria Transformation – negative DNA taken upTransduction by bacteriophages or other virusesMutation

ALL of these introduce GENETIC VARIATION!

Nucleic Acid Hybridization

• Used to detect genes

• The DNA of the cell is denatured to produce single stranded DNA.

• The radioactive probe will hybridize (bond) with complementary bases if present.

• Probes can be radioactive isotopes or

flourescent dyes.

The radioactive probe is made by determining a short segment of the protein sequence, then "back translating" to the possible short DNA sequences called oligomers.

Then these DNA oligomers (known as "oligos") are radiolabeled, and applied to the blotted clones. 

They should hybridize only to clones containing sequence encoding the desired protein.

How does this work?• Protein segment

gly – gly – ser – glutamic acid

• Look on Genetic Codon chart, find mRNA codons

GGU – GGU – UCU – GAA

• Make a radioactive DNA

(oligos)probe

CCA – CCA – AGA - CTT

Expression of eukaryoticgenes in prokaryotes

• Use an expression vector with a prokaryotic promoter upstream from the location of the gene (ie operon)

• Create artificial genes without introns since bacteria do not have the machinery for eliminating introns.

• YACS

What are YACS?

• Yeast artificial chromosomes that carry foreign DNA.

• Yeast cells have plasmids that can act as vectors.

Electroporation

• injecting DNA into eukaryotic cells

PCR Polymerase Chain Reaction

• Used to amplify DNA

• Discovered by Kary Mullis (GT grad)

A Thermocycler

Steps of PCR?

• Denature DNA (94-96 C)

• Anneal (base pair) primers (50 – 65 C)

• Extend primers (72 for polymerase to work)

• Machines called thermocyclers do this.

http://www.dnalc.org/ddnalc/resources/shockwave/pcranwhole.html

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/polymerase_chain_reaction.html

Fig. 20-8b

Cycle 1yields

2molecules

Denaturation

Annealing

Extension

Primers

Newnucleo-tides

3 5

3

2

5 31

Fig. 20-85

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

• In PCR, a heat-stable DNA polymerase is used, most commonly Taq Polymerase from the thermophilic microbe Thermus aquaticus. 

• Thomas Brock discovered T. aquaticus  from a hot spring at Yellowstone National Park.

Applications of PCR

PCR has replaced cloning for many purposes, particularly the sequencing of DNA. 

• It is faster and requires no vectors, which can mutate as they reproduce.  

• It can be used forensically, to amplify tiny amounts of DNA from criminal evidence; or clinically, to detect DNA sequences linked to inherited disorders.  

What is gel electrophoresis?

• A technique to separate DNA based on the movement of DNA fragments from neg to pos (DNA is neg).

• Smaller fragments travel farther.

• Samples are placed in gels.

Gel Electrophoresis

Southern Blotting“DNA Fingerprinting”- named for Edwin Southern- used to identify DNA fragments

1. Isolate DNA2.Cut DNA into fragments with restriction enzymes.3.Electrophorese.4.Blot onto nylon membrane.5.Apply radioactive probes.6.Wash to remove unbonded probes.

http://highered.mcgraw-hill.com/sites/0072556781/student_view0/chapter14/animation_quiz_5.html

In DNA fingerprinting

• Single nucleotide polymorphisms (SNPs) are useful genetic markers

• These are single base-pair sites that vary in a population (most of our DNA is identical

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/restriction_fragment_length_polymorphisms.html

Fig. 20-21

Disease-causingallele

DNA

SNP

Normal alleleT

C

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/video_quiz_-_world_trade_center_dna.html

Many genetic diseases are the result of a

polymorphism at a single locus.

• When a restriction enzyme is added, SNPs result in DNA fragments with different lengths, or restriction fragment length polymorphisms (RFLP)

• Some polymorphisms cause disease, while some do not. Others indicate a predisposition to disease.

Hemoglobin S is missing a restriction site due to a polymorphism

RFLP Analysis in Paternity Cases

RFLP Analysis in Paternity Cases

Short Tandem Repeats• Even more sensitive is the use of

genetic markers called short tandem repeats (STRs), which are variations in the number of repeats of specific DNA sequences

• The Human Genome Project has shown that there are tens of thousands of STR loci in human DNA.

• An individual inherits one copy of an STR from each parent,

D7S280 is one of the 13 core CODIS STR genetic loci. This DNA is found on human chromosome 7. The DNA sequence of a representative allele of this locus is shown below. The tetrameric repeat sequence of D7S280 is "gata". Different alleles of this locus have from 6 to 15 tandem repeats of the "gata" sequence. How many tetrameric repeats are present in the DNA sequence shown below?

1 aatttttgta ttttttttag agacggggtt tcaccatgtt ggtcaggctg actatggagt 61 tattttaagg ttaatatata taaagggtat gatagaacac ttgtcatagt ttagaacgaa121 ctaacgatag atagatagat agatagatag atagatagat agatagatag atagacagat181 tgatagtttt tttttatctc actaaatagt ctatagtaaa catttaatta ccaatatttg241 gtgcaattct gtcaatgagg ataaatgtgg aatcgttata attcttaaga atatatattc301 cctctgagtt tttgatacct cagattttaa ggcc

If the genotypes of both parents are known, we use a Punnett Square to predict the possible phenotypes of their offspring. Each child inherits one allele of a given locus from each parent. Panel (a) - At the D21S11 locus, the children of Bob Blackett and wife

Anne can have four different genotypes. Son David is 28, 31. Daughter Katie is 29, 30. Panel (b) - Bob Blackett inherited the 31 allele from his mother, Norma. Therefore the 29 allele is paternal.

If Bob's paternal was not 29, what would be your conclusion?

Huntington’s Disease can be diagnosed by the number of CAG repeats

The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington's

disease. The male parent, as shown by the black box, got Huntington's disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's disease, and the age at which the symptoms first began is shown by the number

above the band from the PCR fragment.

What is the prognosis for

the normal children 4, 6,

and 9?

Sanger Sequencing

• Used to sequence short segments of DNA• Single-stranded fragments are incubated

with fluorescent-tagged short segments for DNA hybridization.

• When fragments hybridize with the tagged nucleotide, the hybridization stops.

• Fragments are electrophoresed and analyzed.

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/sanger_sequencing.html

Analyzing Expression of Genes

• Northern Blotting – using radioactive probes to look for mRNA being produced

• RT-PCR – Reverse transcriptase-polylmerase chain reaction – makes cDNA from mRNAs and then PCRs the DNA for electrophoresis

• in situ hybridization – can locate specific mRNA’s in cells

• Micro – arrays - Isolate mRNA from cells, make cDNA using reverse transcriptase, then uses cDNA to explore collections of genomic DNA to see if they hybridize

http://glencoe.mcgraw-hill.com/sites/9834092339/student_view0/chapter18/using_a_dna_microarray.html

• Microarrays are useful in discerning gene expression in different tissues AND at different stages of development.

• Different brightness and

colors signify rates of

expression.

Google Image Result for http://www.g2conline.info/content/1178/1

178_what_microarray_thumb.jpg

http://www.dnalc.org/resources/3d/26-microarray.html

An example

DNA Microarray Methodology Animation

Determining Gene Function

• In vitro mutagenesis – changes made to cloned gene, gene returned to cell and it “knocks out” the normal gene. Then look for abnormalities.

• RNA interference (RNAi) – uses RNA to block translation of mRNA and see what happens.

Cloning Organisms

• Organismal cloning – producing genetically identical individuals from a single somatic cell of a multicellular organism

In plants• Steward demonstrated

genomic equivalence in plants by growing carrot plants from differentiated root cells.

• Most plant cells remain totipotent, retaining the ability to give rise to a complete new organisms.

In Animals

• Briggs and all transplanted nuclei from embryonic frog cells into enucleated egg cells and produced cloned frogs

• Nuclear transplantation – name of process

• Whether normal development occurred depended on developmental age of the transplanted nucleus.

Fig. 20-17

EXPERIMENT

Less differ-entiated cell

RESULTS

Frog embryo Frog egg cell

UV

Donornucleustrans-planted

Frog tadpole

Enucleated egg cell

Egg with donor nucleus activated to begin

development

Fully differ-entiated(intestinal) cell

Donor nucleus trans-planted

Most developinto tadpoles

Most stop developingbefore tadpole stage

Nuclear Transplantation

And then Dolly came along in 1997

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

Why Dolly died young 6 yrs

• Dolly's telomeres were found to be approximately 80% of the length they should be for a sheep her age.

• Also there is the concern of damaged DNA being carried into the clone

Cloned animals do not look exactly like the transplanted nucleus due to cytoplasmic affects.

RainbowCC

CC and her Surrogate mom

Hi Mrs. Smith!

• In most nuclear transplantation studies, only a small percentage of cloned embryos have developed normally to birth

• Many epigenetic changes, such as acetylation of histones or methylation of DNA, must be reversed in the nucleus from a donor animal in order for genes to be expressed or repressed appropriately for early stages of development

Stem Cells

• Relatively unspecialized cells that continue to reproduce themselves and can be induced to form specialized cells

• Embryonic cells are more totipotent than adult stem cells

http://cbm.msoe.edu/stupro/so/SOStemCellVideo2010.html

• Therapeutic cloning – using stem cells to replace organs and tissues

• Reproductive cloning – using stem cells to reproduce new organisms

• Both raise ethical

debates

Research points to a new direction in Stem Cell Research

InducedPluripotentStem Cells

The Nobel Prize in Medicine 2012 was awarded to two biologists for their breakthroughs in the field of stem-cell research — two discoveries that happened 44 years apart. The honors go to Britain's Sir John B. Gurdon and Japan's Shinya Yamanaka for their pioneering work with the life-shaping cells, which can be reprogrammed to create any kind of tissue in the body.

Benefits of DNA technology

• Medical Applications

• identification of human genes in which mutation plays a role in genetic diseases

Huntington’s Disease can be diagnosed by the number of CAG repeats

The data below shows the results of electrophoresis of PCR fragments amplified using probes for the site which has been shown to be altered in Huntington's

disease. The male parent, as shown by the black box, got Huntington's disease when he was 40 years old. His children include 6 (3,5,7,8,10,11) with Huntington's disease, and the age at which the symptoms first began is shown by the number

above the band from the PCR fragment.

What is the prognosis for

the normal children 4, 6,

and 9?

Human Gene Therapy

• Gene therapy is the alteration of an afflicted individual’s genes

• Vectors, such as viruses, are used for delivery of genes into specific types of cells, for example bone marrow

• It may be difficult to target cells.• Gene therapy raises ethical questions,

such as whether human germ-line cells should be treated to correct the defect in future generations

Fig. 20-22

Bonemarrow

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

Pharmaceutical Products

• Advances in DNA technology and genetic research are important to the development of new drugs to treat diseases

• In particular “pharm” animals and plants can be used to produce certain products

Fig. 20-23

Forensic Evidence and Genetic Profiles

• An individual’s unique DNA sequence, or genetic profile, can be obtained by analysis of tissue or body fluids

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

Environmental Cleanup

• Some modified microorganisms can be used to extract minerals from the environment or degrade potentially toxic waste materials

• Biofuels make use of crops such as corn, soybeans, and cassava to replace fossil fuels

Genetic Engineering in Plants

• Agricultural scientists have endowed a number of crop plants with genes for desirable traits

• The Ti plasmid is the most commonly used vector for introducing new genes into plant cells

Is this safe?

• Most public concern about possible hazards centers on genetically modified (GM) organisms used as food

• Some are concerned about the creation of “super weeds” from the transfer of genes from GM crops to their wild relatives

Fig. 20-25

Site whererestrictionenzyme cuts

T DNA

Plant with new trait

Tiplasmid

Agrobacterium tumefaciens

DNA withthe geneof interest

RecombinantTi plasmid

TECHNIQUE

RESULTS

• Guidelines are in place in the United States and other countries to ensure safe practices for recombinant DNA technology