genetic engineering · 2018. 9. 5. · genetic engineering is a process in which recombinant dna...

Genetic Engineering

• Genetic Engineers can alter the DNA code of living organisms.

• Selective Breeding

• Recombinant DNA

• PCR

• Gel Electrophoresis

• Transgenic Organisms

Selective Breeding

• Breed only those plants or animals with desirable traits

• People have been using selective breeding for 1000’s of years with farm crops and domesticated animals.

KEY CONCEPT

DNA sequences of organisms can be changed.

Genetic EngineeringGenetic engineering is a process in which recombinant DNA (rDNA) technology is used to introduce desirable traits into organisms. fda.gov

http://www.fda.gov/AnimalVeterinary/DevelopmentApprovalProcess/GeneticEngineering/

Recombinant DNA

• The ability to combine the DNA of one organism with the DNA of another organism.

• Recombinant DNA technology was first used in the 1970’s with bacteria.

Recombinant Bacteria1. Remove bacterial DNA (plasmid).

2. Cut the Bacterial DNA with “restriction enzymes”.

3. Cut the DNA from another organism with “restriction enzymes”.

4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria.

5. Reproduce the recombinant bacteria.

6. The foreign genes will be expressed in the bacteria.

Benefits of Recombinant Bacteria

1. Bacteria can make human insulin or human growth hormone.

2. Bacteria can be engineered to “eat” oil spills.

Recombinant DNA Technology

Manipulating DNA: Ingredients

• Restriction enzymes (a.k.a. restriction endonucleases)

– Cut DNA

• Ligase

– Joins DNA strands together

• Plasmid DNA

– Circular bacterial DNA

bacteria

• Has helped develop biotechnology and genetic engineering

• Fast growth rate

• diversity

Bacterial DNA

• Not enclosed in nucleus

• Have 1 chromosome –large circular loop

• Also include smaller loops of independent DNA - plasmids

plasmids

• Extra-chromosomal DNA

• Small freewheeling circles of DNA/genetic material

• Can readily pass from one cell to another

• Can be used to transfer genes between species

v

v

transformation

• Process of genetic material transfer in bacteria

• DNA is released by bacteria into the surroundings (medium) and then taken up and incorporated into the DNA by another/nearby bacteria

Transformation

• One of the basic principles of genetic engineering

Viruses and gene transfer

Viruses

• occupy borderline between living and non living

• DNA or RNA housed in protective protein coats

• Contain genetic information to make copies of themselves

• Lack biochemical machinery to carry out own replication


Bacteriophages (phages)

• viruses that infect bacteria

• Settle on bacterial host and inject their DNA into cells

• Information encoded in viral DNA dictate bacterial cell to make new virus parts (DNA and protein)

• Result in newly constructed viruses


• Viruses can be thought of as the first genetic engineers

• Can modify cells of other species to carry out their own genetic instructions

• DNA that has been created artificially

• from two or more sources incorporated into a single molecule

• 1st requirement: small DNA fragments

• DNA snippers ( restriction endonucleases, REs)

Recombinant DNA

Recombinant DNA

Restriction endonucleases

• restriction enzymes

• Snips DNA molecules at particular sequence of nucleotides

• Different enzymes recognize and cut different sequences

• Can produce standard DNA fragment with known sequence at cut ends

Recombinant DNA: restriction enzymes

Recombinant DNA: cut and paste

• Any 2 DNA cut by the same restriction enzyme can be joined together

• Complementary sequences on sticky ends (tails)

• Binding is weak – can be broken by heat

• Connection made more secure with Ligase

Putting new genes into cells• Recombinant DNA can be

introduced into host cell

• rDNA usually contain gene not found in host cell

• Introduction of rDNA modifies host cell in some way

• Example: introduce gene producing antiviral protein into bacteria

• Challenge: get rDNA into host cells without disrupting normal function

Making antibiotic resistant bacteria + toad DNA

Production of human interferon from bacteria

Gene expression and control• Presence of gene can make cell carry out genetic instructions

– not always the case

Think..........

• All cell in your body contains same amount of genes

• But cells are not alike

• Genetic potential is not the same as genetic fate

• Only a proportion of genes are “turned on” or expressed at one time

• Results in unique properties of cells

Gene expression and control

• Regulatory genes - Switch mechanism that control gene expression

• Structural genes – encode proteins

• Regulatory genes promote or inhibit expression of structural genes

• Promoter region located adjacent to structural genes

• Switches that control gene expression should be included when transplanting genes to make protein products

Entire organisms can be cloned.• A clone is a genetically identical copy of a gene or of

an organism.

• Cloning occurs in nature.

– bacteria (binary fission)

– some plants (from roots)

– some simple animals (budding, regeneration)

• Mammals can be cloned through a process called nuclear

transfer.

– nucleus is removed from an egg cell

– nucleus of a cell from the animal to be cloned is

implanted in the egg

• Cloning has potential benefits.

– organs for transplant into humans

– save endangered species

• Cloning raises concerns.

– low success rate

– clones “imperfect” and less healthy than original animal

– decreased biodiversity

Clones• identical genetic copy of either a piece of DNA, a cell, or a whole

organism

Cloning from plant cells

Cloning animals: nuclear transfer

Dolly Cc (Copy cat)

Noah

Cloning cells

• plasmids most commonly used in recombinant DNA technology replicate in E. coli

• engineered to optimize their use as vectors in DNA cloning

• contain little more than the essential nucleotide sequences required for their use in DNA cloning

Cloning DNA

• Multiple cloning site (MCS)

• Origin of replication (ORI)

• Antibiotic resistance gene

Making recombinant DNA

Selection of transformants

Genetic engineering:

producing recombinant protein

DNA binding protein

DNA binding protein GFP Recombinant protein

DNA binding protein



DNA binding protein GFP



DNA binding protein GFP

DNA binding protein

Genetic engineering: Bt corn

http://fig

.cox.miami.edu/~cmallery/150/gene/c7.20.4a.cloning.jpg

The DNA of plants and animalscan also be altered.

PLANTS

1. disease-resistant and insect-resistant crops

2. Hardier fruit

3. 70-75% of food in supermarket is genetically modified.

How to Create a Genetically Modified Plant

1.Create recombinant bacteria with desired gene.

2. Allow the bacteria to “infect" the plant cells.

3. Desired gene is inserted into plant chromosomes.

Genetically modified organisms are called

transgenic organisms.

TRANSGENIC ANIMALS

1. Mice – used to study human immune system

2. Chickens – more resistant to infections

3. Cows – increase milk supply and leaner meat

4. Goats, sheep and pigs – produce human proteins in their milk

Human DNA in

a Goat Cell

This goat contains a human

gene that codes for a blood

clotting agent. The blood

clotting agent can be harvested

in the goat’s milk.

.

Transgenic Goat

Gel Electrophoresis

• This technology allows scientists to identify someone’s DNA!

Steps Involved in Gel Electrophoresis

1. “Cut” DNA sample with restriction enzymes.

2. Run the DNA fragments through a gel.

3. Bands will form in the gel.

4. Everyone’s DNA bands are unique and can be used to identify a person.

5. DNA bands are like “genetic fingerprints”.

http://www.dnalc.org/ddnalc/resources/electrophoresis.html

New genes can be added to an organism’s DNA.

• Genetic engineering involves changing an organism’s DNA to give it new traits.

• Genetic engineering is based on the use of recombinant DNA.

• Recombinant DNA contains genes from more than one organism.

(bacterial DNA)

• Bacterial plasmids are often used to make

recombinant DNA.

– plasmids are loops of

DNA in bacteria

– restriction enzymes cut

plasmid and foreign DNA

– foreign gene inserted into

plasmid

Genetic engineering produces organisms with new traits.

• A transgenic organism has one or more genes from another organism inserted into its genome.

• Transgenic bacteria can be used to produce human

proteins.

– gene inserted into plasmid

– plasmid inserted into bacteria

– bacteria express the gene

• Transgenic plants are common in agriculture.

– transgenic bacteria

infect a plant

– plant expresses

foreign gene

– many crops are now

genetically modified

(GM)

• Transgenic animals are used to study diseases and

gene functions.

– transgenic mice used to study development and

disease

– gene knockout mice used to study gene function

• Scientists have concerns about some uses of genetic

engineering.

– possible long-term health effects of eating GM foods

– possible effects of GM plants on ecosystems and

biodiversity

biotechnology techniques

• Based on naturally occurring properties of different cells, genes, and enzymes

• Adopted by researchers for their own purposes

Manipulating DNA

Polymerase Chain Reaction

• “Kary B. Mullis, working for Cetus Corporation, developed the Polymerase Chain Reaction in 1983.

• The technique allows for the rapid synthesis of DNA fragments. In about an hour, over 1 million copies of a DNA strand can be made.

• Mullis left Cetus in 1986. For his development of PCR, he was co-awarded the Nobel Prize in chemistry in 1993. Mullis is currently doing HIV and AIDS research

• The technique has been invaluable to the development of biotechnology and genetic engineering. Its real world applications include gene mapping and forensic science.”

• library.thinkquest.org

Kary Mullis: Surfer-Scientist

www.revolt.it

http://www.revolt.it/

Polymerase Chain ReactionPCR

• PCR allows scientists to make many copies of a piece of DNA.

1. Heat the DNA so it “unzips”.

2. Add the complementary nitrogenous bases.

3. Allow DNA to cool so the complementary strands can “zip” together.

http://www.sumanasinc.com/webcontent/animations/content/pcr.html

Polymerase Chain Reaction

PCR

library.thinkquest.org

DNA Sequencing

• Refers to determining the order of nucleotide (G, A, T, and C) in a stretch of DNA

Human Genome Project was initiated1986

2003Human Genome Project was completed (2 years earlier)

17 YearsHuman Genome Project

A C T G AT C G AT C G TA AT C TA C G G TAT G A C

1st Generation: Chain termination Method

• Sanger Method

DNA elongation by Polymerase chain reaction (PCR) are terminated by addition of modified nucleotides (Sanger et.al. 1977)

Primer

Template

dATP dCTP dGTP dTTP

DNA Polymerase

Ch

ain Elo

ngatio

n

ddATP ddCTP ddGTP ddTTP

Ch

ain Term

inatio

n

AT

CG

Gel Electrophoresis

Visu

alization

A C T G AT C G AT C G TA AT C TA C G G TA

1st Generation: Chain termination Method

• Sanger Method

DNA elongation by Polymerase chain reaction (PCR) are terminated by addition of modified nucleotides (Sanger et.al. 1977)

dATP dCTP dGTP dTTP

DNA Polymerase

Ch

ain Elo

ngatio

n

T G A C T AGC T AGC AT

ddATP

TA*

ddATP ddCTP ddGTP ddTTP

Ch

ain Term

inatio

n

TA G C TA G C AT TA G AT G C C AT *

1st Generation: Chain termination Method• Visualization

Agarose Gel Electrophoresis acts as a sieve that separates the DNA fragments into differents sizes as they pass through an electric current (I).

Gel Electro

ph

oresis

ddATP ddCTP ddGTPddTTP

1st Generation: Chain termination Method• Visualization

Agarose Gel Electrophoresis acts as a sieve that separates the DNA fragments into differents sizes as they pass through an electric current (I).

Gel Electro

ph

oresis

ddATP ddCTP ddGTPddTTP

I

+

-

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

T G A C T *

Sequencing DNA: Reading the sequence using a computer

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

Sequencing the Human Genome Using a Shotgun

http://seqcore.brcf.med.umich.edu/doc/educ/dnapr/sequencing.html

1st Generation:Capillary Sequencing

ABI 3730xl DNA AnalyzerSource: http://www2.jabsom.hawaii.edu/genomicscorefacility/sequencings.html


• ABI 3730xl DNA Analyzer

• Sanger method

• 96 capillaries, 36-50cm, 50um ID

• Fluorescent label terminal nucleotides

• Allow single lane per sample

Source: http://www2.jabsom.hawaii.edu/genomicscorefacility/sequencings.html


• ABI 3730xl DNA Analyzer

• 400-900bp read length

• Minimum DNA:40ng/uL per amplicon

• Cost:

Php 250.00 / capillary

http://seqcore.brcf.med.umich.edu/doc/dnaseq/fair3730.gif

SECOND GENERATION SEQUENCINGMassive Parallel Sequencing Reactions due to micro- and nano-technology advances reducing size of sample components and reagent costs

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SECOND GENERATION SEQUENCINGMassive Parallel Sequencing Reactions due to micro- and nano-technology advances reducing size of sample components and reagent costs

Second Generation Sequencing:Automate Clonal Amplification

• EMULSION PCR • BRDIGE PCR


• EMULSION PCR • Fragments, with adaptors, are PCR amplified in water or oil drops

• One primer is attached to the surface of the bead

• DNA molecules are synthesized on the beads from a single DNA fragment

• Used by Roche 454, IonTorrentand SOLiD


• BRDIGE PCR

• Fragments are flanked with adaptors

• A flat surface (chip) coated with two types of primers, corresponding to the adaptors

• Amplification proceeds in cycles, with one end of each bridgetethered to the surface

• Clusters of DNA molecules are generated on chip with each cluster originated from single DNA fragment

• Used by Illumina

Second Generation Sequencing:Nucleotide Detection

PYROSEQUENCINGSEQUENCINGBY LIGATION

SEMICONDUCTORSEQUENCING

CYCLIC REVERSIBLETERMINATION

REAL TIMESEQUENCING


• Pyrosequencing• Single nucleotide

addition• Substrate addition

– Luciferin, adenosine 5’ phosphosulfate

– Pyrophosphate release + Luciferin Drive light production

• Apyrase wash for next turn


• Sequencing by LigationSequencing is performed with a LIGASE, rather than a polymerase

For each cycle

• Introduction of partially degenerate population of fluorescently labeled octamers

• Label correlates with the central 2 bp (XX or xy) in the octamer (2-base coding)

• Imaging in four channels

• Labeled portion of the octamer (zzz) is cleaved via a modified linkage between 5 and 6


• Reversible Terminators by Illumina

• Simultaneous addition of the 4 modified dNTPs (with labels/dyes) and removable stopper in 3’ end

• Modified Polymerase drives synchronous extension

• Imaging detected in four channels– Signal in channels detects

which nucleotide was incorporated


• REAL-time Sequencing (PacBio Sciences)• No termination of DNA synthesis

• Uses highly efficient phi29 DNA polymerase

• Images release of dyes phospholinked in nucleotides being incorporated


• Semiconductor Sequencing

• Single nucleotide addition per cycle

• Nucleotide incorporation results to release of H+

– Detected by Ion/pH Sensor

• Non-visual detection

Second Generation Sequencing

WHOLE GENOME SEQUENCING

Determining the entire genome of an organism, either de novo or through mapping.

TARGETED SEQUENCING

Selected regions of DNA are enriched prior to sequencing to achieve the high coverage necessary for calling variants such as SNPs, insertions, and deletions.

Second Generation Sequencing

METAGENOMIC SEQUENCING

DNA is extracted from environmental samples and is then enriched for sequencing of microbial genomic markers such as 16s rRNA genes.

RNA SEQUENCING

mRNA is selectively extracted through poly-A capture, from which cDNA libraries are generated for sequencing.

genetic engineering · 2018. 9. 5. · genetic engineering is a process in which recombinant dna...

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