Genetic Technology

Manipulating Genes

A. Genetic Engineering

Genetic engineering (AKA recombinant DNA technology) is faster & more reliable method of selecting certain trait in population

• Artificial selection is done by humans breeding specific individuals with certain traits - slow

• Natural selection is nature selecting specific individuals with certain traits – very slow

• Genetic engineering involves cleaving (cutting out) DNA from one organism into small fragments & inserting desired gene into host DNA of same or different species

Desired gene

Host DNA

Also called recombinant DNA technology since DNA gets recombined to make one new one

If plants or animals contain foreign DNA from this technology, are called transgenic organisms or genetically modified organism (GMO)• Example is tobacco plant that contains

glowing gene from firefly – plant glows!!

Example of Transgenic Organisms

Tobacco PlantCaterpillar

Zebra Fish

Firefly Bioluminescence

Making transgenic or GMO takes 3 steps:1. cleave DNA – isolate DNA fragment

2. vector - attach DNA fragment to carrier

3. insertion - insert DNA into host organism

B. Steps of Engineering

1. Cleave DNA

2. Make a vector

3. Insert into host

1. DNA Cleavage Must isolate small parts of DNA (DNA can

contain millions of base pairs• use special enzymes called restriction

enzymes that cut both sides of dsDNA at specific areas of nucleotide sequence

• depending on which way DNA is cut, get 2 different ends: o sticky endo blunt end

o sticky end dsDNA is cut leaving some single strands can only do that if there is a palindrome = letter

order written same way backwards as forwards ex: “mom” backwards is “mom” but with dsDNA, both

sides are included “GAATTC” on top side (forwards) and “CTTAAG” on

bottom side (backwards)

A T C C A G G A A T T C C A A G C T CT A G G T C C T T A A G G T T C G A G

restriction enzyme recognizes specific palindromes and will cut somewhere within there ex: EcoRI recognizes GAATTC & will cut in b/t G – A on both sides leaving sticky ends ready to bond

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

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

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

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

T A G G T C C T T A A

After Cleaving

TTAA and AATT sticky end have nothing to bond to, so if same restriction enzyme cuts DNA of organism and host’s organism, both sticky ends will match so bonding will be easier

Example of Sticky ends

o blunt end DNA is cut all way through like with

scissors

A T C C A G G A C T T C C A A G C T CT A G G T C C T G A A G G T T C G A G

A T C C A G G A C T T C C A A G C T CT A G G T C C T G A A G G T T C G A G

both ends are bonded with other bases so are blunt

After Cleaving

Examples of Restriction Enzymes

2. Attach to vector Loose fragments of DNA

need to be attached to vector (carrier) first

Two types of vectors:o Biological vector: bacteria

plasmid or viruso Mechanical vector:

micropipette or microscopic metal bullet

Since both DNA and vector were cleaved with same restriction enzyme, both ends will match • Join pieces using DNA ligase

3. Insertion into host Recombined plasmid (or other vector) is

inserted into host’s cell When host replicates, inserted DNA also

replicates producing more of that desired gene• bacterial plasmid can replicate every 20

min!

o Bacterial plasmid Inserts plasmid into

bacteria’s cytoplasm

o Virus Injects DNA directly

into host’s DNA Process called

transduction

Plasmid can replicate 500 times per cell, and each clone replicates 500 times… and so on• Clone: genetically identical copies of

original

Dolly (1996-2003)The first ever

cloned animal

can also replicate DNA segments by using Polymerase Chain Reaction (PCR)

• dsDNA strands are separated (unzipped) by heat

• special heat-resistant enzymes replicate DNA

• important advancement technique used to match DNA with very little DNA to begin withDon’t need much DNA from crime

scenes

PCR Technique

Example of Recombinant DNA

C. Uses for Genetic Engineering

recombinant DNA (genetic engineering) is currently useful in many areas of life

1. Industry

2. Medicine

3. Agriculture

a. clothing: bacteria E. coli are transgenic with DNA to make indigo dye indigo dye in nature is VERY

expensive, so can make blue jeans cheaply

b. food: making corn that has high protein content (corn is mostly carbohydrate)

c. fuel: use corn husks to make fuel for cars

d. sewage: clean water using bacteria

1. Industry

a. hormone: can produce human growth hormone (hGH) to treat people with growth disorders (Achondroplasia, Turner syndrome)

b. medicine: produce human insulin (formerly bovine/cow) with bacterial plasmids

c. diseases: transgenic sheep are produced that produce Factor VIII protein for hemophiliacs

d. Vaccines: remove virus’ dangerous genes

2. Medicine

making more/bigger/healthier/fresher food Crops resistant to viruses and insects canola plants make more canola oil peanuts & soybeans that don’t cause

allergic reactions corn that can grow with very little water

(survive drought)

3. Agriculture

D. The Human Genome Project

International effort started in1990, Human Genome Project (HGP) was organized to completely map and sequence human genome• complete sequence of nucleotides

(3.2 billion) in human DNA (completed 2000).

• Complete map of 20,000 genes (2006) on 23 sets of chromosomes

Human Genome Project

Leaders of the Genome Project (Dr. Landers and Dr. Collins

1. How did they find out that genes U-Z are on chromosome set #2 and not on set #8?

2. How do we know gene N is next to M and O and not somewhere else?

ANSWER: Linkage Maps

ABCD

ABCD

UVWXYZ

UVWXYZ

Set# 2LMNOP

Set# 8LMNOP

QRS

QRS

How did they do that?

Linkage map: genetic map that shows relative locations of genes on a chromosome

• Found locations of genes on specific chromosomes, but didn’t know the order

o Gene M is on chromosome 11o Where on chromosome 11 is gene M?

• Can find RELATIVE order of genes from a linkage map

Linkage Map

Linkage Map

Chromosome 11

Chromosome M

Using PCR, can make millions of copies of DNA fragments to find patterns in certain geneso Use genetic markers to trace inheritance of

genes, which shows us where that gene is located relative to the others

• Father has genes M & HD

• Mother does not Out of 5 kids, 3

inherited M, and of those 3, 2 also got HD

M & HD are close to each other

Gel Electrophoresis

Process of separating DNA fragments to compare sizes and therefore similarities• Electricity is sent through gel containing DNA fragments• DNA pieces will migrate toward bottom

o Smaller pieces will “run” fastero Larger pieces will be stuck toward top

large

small

Genetic Markers

Paternity Tests

Results: D2 not Dad’sResults: S2 adopted

sequencing human genome compares DNA fragments to each other• pieces that overlap are pieced together

How did they sequence it?

ABCD NOPQ DEFGHIJ

IJKLMNOP RSTUVWX WXYZ BCD

LMNOPQRS

All these fragments came from cleaving DNA into little workable pieces

ABCDEFGHIJKLMNOPQRSTUVWXYZ

By lining up pieces that overlap, can get entire sequence

Diagnosing genetic disorders – individuals find out if they are carrying gene for specific disease Can be done for fetuses using epithelial cells (from

amniotic fluid) Dilemma – do YOU want to know if you have gene

for cancer or heart disease? Pros: can alter lifestyle NOW to help prevent

cancer or heart disease from coming Cons: always in fear about what may or may

not happen

E. Applications of HGP

Gene therapy – inserting normal genes into human cells to correct genetic disorder Cystic fibrosis, sickle-cell anemia, hemophilia,

AIDS, cancer, heart disease are all being studied as genetic diseases in which gene therapy may work

Gene Therapy

DNA fingerprinting – compare unknown DNA to known DNA to find out if they match DNA cut by restriction enzymes would show same sizes

each time (same palindrome sequence) Called restriction fragment length polymorphisms

(RFLPs)o Solve crimeso Maternity/paternity

DNA Fingerprinting

Paternity Tests

Results: D2 not Dad’sResults: S2 adopted