Chapter 12- DNA Technology and the Human Genome

• Biotechnology• Blunt ends• cDNA• Conjugation• DNA fingerprinting• DNA ligase• DNA microarrays• F factor• Gel electrophoresis• Gene cloning• Gene therapy• Genetic marker• Genomic library• Genomics• GM organisms• Golden rice• Human Genome Project

• PCR• Plasmid• Probe• R plasmid• Recombinant DNA • Restriction enzymes• Restriction fragments• Sex pili• Sticky ends• Telomeres• Ti plasmid• Transduction• Transformation• Transgenic organism• Transposons• Vaccine• Vector

Bacterial mating shows how genes are transferred

• Asexual reproduction

• 3 ways to transfer DNA:– 1-Transformation- DNA is taken in from

surrounding fluid• Explains Griffiths experiment

– 2-Transduction- phage transfers bacterial genes

• Has genes from previous host

– Conjugation- cells join and a copy of DNA is transferred via mating bridge

– Figures 12.1 A,B, and C in text pg 232

Plasmids in conjugation

• Small, circular piece of DNA separate from bacterial chromosome– Carry genes during conjugation

• F-factor- piece of DNA that carries genes for sex pilli and other genes necessary for conjugation– Behaves 2 ways during conjugation:– 1- it is integrated into bacterial chromosome– 2- it is a plasmid

• When it carries genes for more than replication and conjugation it is acting as a vector

• R –plasmids- “resistance” – carry genes for enzymes that destroy antibiotics– They can also carry genes of interest and that gene can then be

transcribed and translated

Plasmids in conjugation

What is biotechnology?

• Using organisms to perform practical tasks– Bacteria making human proteins (insulin,

HGH)– Genes for pest resistance put into plants– Bacteria with genes to break down toxic waste– Golden Rice

How is this transformation done?• Restriction enzymes- recognize short nucleotide

sequences and cut at a specific point– Used in nature to chop of intruder DNA to protect – Cuts ends in two ways: sticky or blunt

• DNA ligase- catalyzes covalent bonds between nucleotides

• Putting genes into a piece of DNA using restriction enzymes and DNA ligase is producing recombinant DNA

• Genes can be cloned using this method

Gene Cloning

• Produces multiple copies of a gene

• Human DNA and bacterial plasmid are cut with same restriction enzyme

• They are mixed and with DNA ligase are recombined

• The plasmid is put back into the bacteria and allowed to reproduce asexually

Uses for recombinant DNA?

• Mass produce gene products– “pharm” animals can produce human genes– Human insulin can be produced by E. coli– Certain proteins can now be produced and

secreted in an animals milk– Rice fortified with certain vitamins

GMO’s• genetically modified organisms

– Organism that has artificially gained 1 or more genes– To: delay ripening, resistance to spoiling, disease,

herbicide, insecticide, add nutrients *Golden rice– Vectors are usually used to introduce genes to plants

(fig 12.18A)• Ti plasmid- from soil bacteria (induces tumors)• Researchers have removed tumor causing

properties but kept ability to transfer DNA– Recombinant organism is genes of another species is

a transgenic organism

Keeping track of these pieces

• Genomic library-– Set of DNA segments, carried on a plasmid or phage,

from an organisms’ genome

Making cDNA (complementary)

• Using reverse transcriptase-– produces DNA from

RNA template

• DNA without introns– can be transcribed and

translated by bacteria

• Bacteria can then make human protein

Using probes

• Short labeled nucleic acid molecule– Used to identify specific gene or DNA sequence– Bacterial colonies are transferred to filter paper– DNA is heated to separate strands– Radioactive probes are added, filter is “developed”– Compare “picture” with culture to find genes

DNA microarrays

• Used to detect the expression of 1000’s of genes at a time– Tiny pieces of

DNA are attached to glass and tested for hybridization with various DNA molecules

Gel Electrophoresis

• Sorts molecules by size and charge

• Samples are added to wells in a gel

• Electric current allows molecule movement

• Small molecules move farther and faster

Genetic Markers and probes are used with gel electrophoresis

• Genetic markers- chromosomal landmarks that can be used to study inheritance– Non-coding sequences- similar in related

individuals– DNA is cut with restriction enzymes, and run

through electrophoresis gel– Probes can then be used to detect specific

sequences

What if the sample of DNA we have it too small to run a gel?

• PCR- – polymerase chain reaction– amplifies amount of DNA in test tube without

using living cells– DNA sample is put into tube with DNA pol,

nucleotides, and other molecules (primers)– Sample is heated to separate strands then

cooled for nucleotides to bind (done over and over again)

Uses for gel electrophoresis and PCR?

• DNA Fingerprinting- uses short repeats and PCR to amplify

Ironically…• Most of your DNA is junk (97%)!!!

– DNA between genes is repetitive sequences– 2 types:– 1- short repeated sequences by centromere

and at ends of chromosomes by telomeres- protect against DNA loss

– 2- 2nd type- long repeated sequences • Scattered around genome• “jumping genes”- transposons- can jump

into middle of gene and disrupt expression

Barbara McClintock

• Worked at Cold Spring Harbor Lab

• Experimented with Indian corn kernel color and transposons

Uses in medicine:– Therapeutic hormones- insulin, HGH– PCR can help find viruses– DNA microarrays- allows treatment to be tailored to a

specific disorder– Vaccines- harmless variant or derivative of a

pathogen (usually bacteria or virus)• Stimulates immune system to develop defenses

against the pathogen• 1 way- produce harmless artificial mutant of

pathogen– Causes fewer side effects

• 2nd way- engineer cells to produce protein that is found on pathogen’s surface

Gene Therapy

• Altering the afflicted individuals genes– New allele can be inserted into somatic cells of

infected tissue– Bone marrow cells- good candidate because they

include stem cells that can differentiate completely– Bone marrow cells that have been infected with gene

carrying virus are injected into afflicted individual– New ideas focus on adding genes to help not fix

• Ex: heart growing new blood vessels

– There are ethical and technical problems

Human Genome Project

• HGP

• Goal to sequence the whole human genome

• They succeeded

• Gene mapping then physical mapping the DNA sequencing

• http://www.ornl.gov/sci/techresources/Human_Genome/home.shtml