1

AP Biology

Biotechnology

and

Genetic Engineering

AP Biology

Biotechnology Today

Genetic Engineering: process of manipulating genes and

genomes

Biotechnology: process of manipulating organisms or their

components for the purpose of making useful products.

Recombinant DNA: DNA that has been artificially made, using

DNA from different sources

eg. Human gene inserted into E.coli

Gene cloning: process by which scientists can product

multiple copies of specific segments of DNA that they can

then work with in the lab

AP Biology

Techniques of Genetic Engineering

Transformation: bacteria takes up plasmid (w/gene of interest)

Gel electrophoresis: used to separate DNA molecules on basis of size and charge using an electrical current (DNA migrates to + pole)

PCR (Polymerase Chain Reaction): amplify (copy) piece of DNA without use of cells

DNA microarray assays: study many genes at same time

AP Biology

Bacteria

Bacteria review

• one-celled prokaryotes

• reproduce by mitosis

• binary fission

• rapid growth

• generation every ~20 minutes

• 108 (100 million) colony overnight!

• dominant form of life on Earth

• incredibly diverse

AP Biology

Bacterial Genome

• Single circular chromosome

• haploid

• naked DNA

• no histone proteins

• ~4 million base pairs

• ~4300 genes

• 1/1000 DNA in eukaryote

AP Biology

Transformation

Bacteria are opportunists

• pick up naked foreign DNA

wherever it may be hanging out

• have surface transport proteins that are

specialized for the uptake of naked DNA

• import bits of chromosomes from

other bacteria

• incorporate the DNA bits into their

own chromosome

• express new genes

• transformation

(form of recombination)

mix heat-killed pathogenic & non-pathogenic bacteria

mice die

2

AP Biology

Plasmids

Small supplemental circles of DNA 5000 - 20,000 base pairs

self-replicating

• carry extra genes

2-30 genes

genes for antibiotic resistance

• can be exchanged between bacteria

bacterial sex!!

rapid evolution

• can be imported from environment

AP Biology

Plasmids

• A way to get genes into bacteria easily

• insert new gene into plasmid

• insert plasmid into bacteria = vector

• bacteria now expresses new gene

• bacteria make new protein

+

transformed

bacteria gene from

other organism

plasmid

cut DNA

recombinant

plasmid

vector

glue DNA

AP Biology

Biotechnology

Plasmids used to insert new genes into bacteria

gene we want

cut DNA

cut plasmid DNA

insert “gene we want” into plasmid...

“glue” together

ligase

like what? …insulin …HGH …lactase

recombinant plasmid AP Biology

Restriction Enzymes (RE)

• restriction endonucleases

• discovered in 1960s

• evolved in bacteria to cut up foreign DNA

“restrict” the action of the attacking organism

protection against viruses & other bacteria

AP Biology

Restriction Enzymes (RE)

radar

racecar

Madam I’m Adam

Able was I ere I saw Elba

a man, a plan, a canal, Panama

Was it a bar or a bat I saw?

go hang a salami I’m a lasagna hog

palindromes

AP Biology

Restriction enzymes (RE)

• Action of enzyme

• cut DNA at specific sequences

restriction site

• symmetrical “palindrome”

• produces protruding ends

sticky ends

• will bind to any complementary DNA

• Many different enzymes

• named after organism they are found in

EcoRI, HindIII, BamHI, SmaI

restriction enzyme animation

CTGAATTCCG

GACTTAAGGC

CTG|AATTCCG

GACTTAA|GGC

3

AP Biology

Restriction enzymes (RE)

Cut DNA at specific sites

leave “sticky ends”

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAA

GTAACGAATTCACGC

TT

CATTGCTTAAGTGCG

AA

restriction enzyme cut site

restriction enzyme cut site

AP Biology

Sticky Ends

Cut other DNA with same enzymes

leave “sticky ends” on both

can glue DNA together at “sticky ends”

GTAACG AATTCACGCTT

CATTGCTTAA GTGCGAA gene

you want

GGACCTG AATTCCGGATA

CCTGGACTTAA GGCCTAT

chromosome want to add

gene to

GGACCTG AATTCACGCTT

CCTGGACTTAA GTGCGAA combined

DNA

AP Biology

Sticky Ends

TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTCA AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGT

gene you want cut sites cut sites

isolated gene sticky ends

chromosome with new gene added

TAACGAATTCTACGAATGGTTACATCGCCGAATTCTACG CATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAGATG

sticky ends together DNA ligase joins the strands Recombinant DNA molecule

GAATTCTACGAATGGTTACATC AGATGCTTACCAATGTAGCGG

TTGTAACGAATTCTACGAATGGTTACATCGCCGAATTC AACATTGCTTAAGATGCTTACCAATGTAGCGGCTTAAG

AP Biology

Making Recombinant DNA (rDNA)

Restriction enzymes

(RE)

cut gene of interest

and

DNA ligase pastes it into

new plasmid

AP Biology

Gene Cloning 1. CUT

Restriction enzymes cut DNA into fragments that can be isolated and separated

- very specific proteins, recognizes and cuts DNA at specific sequence into pieces

Ex: EcoRI – cuts DNA whenever C-T-T-A-A-G sequence occurs

2. PASTE Production of recombinant DNA (rDNA)

- DNA composed of fragments of DNA

segments from at least two different organisms

- restriction enzymes cut bacterial plasmids (extra circular DNA molecules in bacteria)

- plasmids have “sticky ends” (unpaired bases)

- original DNA is attached to plasmid sticky ends

- ligase stitches rDNA together AP Biology

Gene Cloning

3. INSERT & COPY Reintroduction of DNA into bacterial (cloning) vector

- recombinant DNA taken up with bacterial DNA and now produced by bacterial cell

- recombinant DNA is isolated and CLONED

- PCR (polymerase chain reaction) induced

- purpose: to make 1000s of recombinant plasmids PCR animation

4. FIND DNA sequencing

- Process of reading exact order of bases in fragment of DNA

- makes it possible for scientists to make sure gene of interest has been cloned gene cloning video

4

AP Biology

Gene Cloning (short version)

grow

bacteria

harvest (purify)

protein

transformed

bacteria

plasmid

gene from

other organism

+

recombinant

plasmid

vector

AP Biology 2005-

2006

Applications of Gene Cloning

AP Biology

Applications of Cloning: Genomic Libraries

Cut up all of nuclear DNA

from many cells of an

organism

restriction enzyme

Clone all fragments into

many plasmids at same

time

“shotgun” cloning

Create a stored collection

of DNA fragments

petri dish has a

collection

of all DNA

fragments from the

organism

AP Biology

Applications of Cloning:

cDNA(complementary DNA) Libraries

Collection of only the coding sequences of expressed genes

• extract mRNA from cells

• reverse transcriptase

RNA DNA

from retroviruses

• clone into plasmid

Applications

need edited DNA for expression in bacteria

Ex: human insulin

animation

AP Biology

Applications of Cloning: Entire Organisms

Nuclear transplantation: nucleus of egg is removed

and replaced with nucleus of body cell

AP Biology

Problems with Reproductive Cloning

• Cloned embryos exhibited various defects

• DNA of fully differentiated cells have epigenetic changes

5

AP Biology

Applications of Cloning: Stem Cells

Stem cells

unspecialized cells- can

reproduce itself indefinitely and

differentiate into other specialized

cells

Types:

- Zygote stem cells:

totipotent (any type of cell)

- Embryonic stem cells:

pluripotent (many cell types)

- Adult stem cells (somatic):

- multipotent (a few cell types)

- induced pluripotent

(re- programmed)

AP Biology

Stem Cells for Disease Treatment

AP Biology

Gel Electrophoresis

A method of separating DNA

fragments of different size in a

gelatin-like material using an

electrical field

DNA is negatively charged

when it’s in an electrical field it

moves toward the positive side

+ –

DNA

“swimming through Jello” AP Biology

DNA moves in an electrical field…

so how does that help you compare DNA

fragments?

size of DNA fragment affects how far it travels

small pieces travel farther

large pieces travel slower & lag behind

Gel Electrophoresis

+ –

DNA

“swimming through Jello”

AP Biology

Gel Electrophoresis

longer fragments

shorter fragments

power source

completed gel

gel

DNA & restriction enzyme

wells

-

+ AP Biology

Southern Blotting

Once banding patterns are apparent they are

transferred to a Southern Blot for identification

6

AP Biology

DNA Fingerprints

Comparing blood samples on

defendant’s clothing to

determine if it belongs to

victim

DNA fingerprinting

comparing DNA banding

pattern between different

individuals

~unique patterns

Bozeman video

Naked science video

AP Biology

Uses: DNA Fingerprinting in Forensics

Comparing DNA sample from crime scene with

suspects & victim

–

+

S1

DNA

S2 S3 V

suspects crime scene sample

AP Biology

RFLPs Restriction Fragment Length Polymorphism

• differences in DNA between individuals

• due to differences in “junk” DNA

change in DNA sequence affects

restriction enzyme “cut” site

creates different fragment sizes &

different band pattern

Restriction fragment length polymorphisms

AP Biology

RFLPs in Forensics

AP Biology

Uses: Evolutionary Relationships

Comparing DNA samples from different organisms to measure evolutionary relationships

–

+

DNA

1 3 2 4 5 1 2 3 4 5

turtle snake rat squirrel fruitfly

AP Biology

Uses: Disease Diagnosis

Comparing normal allele to disease allele

chromosome with

disease-causing

allele 2

chromosome

with normal

allele 1 –

+

DNA

Example: test for Huntington’s disease

7

AP Biology

Uses: Paternity Tests

AP Biology

Polymerase Chain Reaction (PCR)

Method for amplifying (making many copies) of a specific segment of DNA

Materials needed:

• template strand from DNA of one cell

• DNA polymerase enzyme

• Nucleotides

• ATP, GTP, CTP, TTP

• Primer (define section

of DNA to be cloned)

AP Biology

Microarray “DNA Chips”

Slide with a sample of

each gene from the

organism

•each spot is one

gene

animation

AP Biology

Applications of Genetic Engineering Gene Therapy Using a Retroviral Vector

AP Biology

Applications of Genetic Engineering

“Pharm” animal: produce human protein secreted in milk for medical use

AP Biology

Applications of Genetic Engineering

STR Analysis (Short Tandem Repeats)

Non-coding DNA has regions with sequences (2-5 base length)

that are repeated

Each person has different # of repeats at different locations

(loci)

Current method of DNA fingerprinting used – only need 20

cells for analysis

8

AP Biology AP Biology

Applications of Genetic Engineering

Medicines and Vaccines

produced by bacteria and

viruses

- E coli: used to make human insulin

- hamster cell cultures: used to make TPA (tissue plasminogen activator)- dissolves blood clots in heart attacks

- EPO (erythropoiten): increase red blood cell production

- interferon: fights viral infections, increases immunity

- vaccines

vaccine animation

AP Biology

Applications of Genetic Engineering Pollution Control

Pollution control

- genetically altered

bacteria used to

decompose garbage

sewage, and

petroleum products

Unlike the left tower, which uses

chemicals, the tower on the right at this

wastewater-treatment plant now uses

bacteria-covered foam blocks (inset) to

eliminate the hydrogen sulfide bubbling

from treated sewage

AP Biology

Applications of Genetic Engineering

Transgenic Organisms

Genetically Modified Organisms (GMOs)

• organisms that contain foreign genes

Transgenesis

The use of recombinant DNA techniques to

introduce new characters (ie. genes) into

organisms (including humans) that were not

present previously.

AP Biology

Applications of Genetic Engineering

GMOs

enabling plants to produce new proteins

Protect crops from insects: BT corn

corn produces a bacterial toxin that kills

corn borer (caterpillar pest of corn)

Extend growing season: fishberries

strawberries with an anti-freezing gene

from flounder

Improve quality of food: golden rice

rice producing vitamin A

improves nutritional value

AP Biology

9

AP Biology AP Biology

Human Genome Project

• U.S government project (1990) – 15 year

timeframe

• goal was to sequence entire human

genome

3 billion base pairs

• Completed in 2003

• Blueprint of a human

• Genomes sequenced thus far*: 58,000 prokaryotes, 2700 eukaryotes, 5300 viruses

AP Biology

Comparison of Genomes

Organism Genome Estimated

Size Genes

Human (Homo sapiens) 3 billion 30,000

Laboratory mouse (M. musculus) 2.6 billion 30,000

Mustard weed (A. thaliana) 100 million 25,000

Roundworm (C. elegans) 97 million 19,000

Fruit fly (D. melanogaster) 137 million 13,000

Yeast (S. cerevisiae) 12.1 million 6,000

Bacterium (E. coli) 4.6 million 3,200

Human Immunodeficiency Virus (HIV) 9700 9

AP Biology

Human DNA

• 3 billion base pairs

• ~20,000 genes

• Only 1.5% codes for

proteins (or RNA)

• Mostly Repetitive DNA:

sequences present in

multiple copies

SNP video

AP Biology

Evolution of Genomes

Interspersed Repetitive DNA

Repetitive DNA is spread throughout genome

• interspersed repetitive DNA (SINEs- Short

INterspersedElements) make up 25-40% of mammalian

genome

• in humans, at least 5% of genome is made of a family of

similar sequences

• 300 bases long

• Aluis an example of a "jumping gene" called a transposon;

AP Biology

Evolution of Genomes

Transposable Elements (jumping genes)

- Make up 75% of repetitive DNA

- DNA sequence that "reproduces" by copying itself &

inserting into new chromosome locations

- Can be moved from one location to another in genome

- Discovered by Barbara McClintock – corn breeding experiments

• 2 Types:

Transposons

Retrotransposons

10

AP Biology

Evolution of Genomes Rearrangements in the Genome

Transposons

•Move within genome via DNA

intermediate

•Insertion of transposon sequence in

new position in genome

•“cut & paste” or “copy & paste” mechanisms

•Requires enzyme transposase

Insertion sequences cause

mutations when they happen to land

within the coding sequence of a

gene or within a DNA region that regulates gene expression

AP Biology

Evolution of Genomes

Retrotransposons

• Most transposons are retrotransposons

• Move within a genome via RNA intermediate,

transcript of the retrotransposon DNA.

• Involves enzyme reverse transcriptase

AP Biology

Evolution of Genomes

Insertion effects of transposons:

Can interrupt or alter gene function

Multiple copies of genes

Duplication genes with related functions

Genes diverge by accumulating mutations

Some become nonfunctional pseudogenes

Eventually, new genes with new functions can occur

AP Biology

Evolution of Genomes

Multigene Families

• Collections of 2 or more identical or very similar genes

• Eg. hemoglobin: -globin and -globin gene families

AP Biology

Evolution of Genomes

Transpositions chromosomal rearrangements

AP Biology

Evolution of Genomes

Transposable elements contribute to evolution

• Promote recombination, disrupt genes or control elements, & carry genes to new locations

• May be harmful or lethal, but can also have small beneficial effects

• Provides raw material for natural selection

• Ex: antifreeze genes in fish (antifreeze proteins- AFP)

• Aids survival in sub zero environments (binds to ice crystals)

• Old protein gene mutates duplicates divergent evolution

HHMI video