1. explain, in general, how restriction enzymes cut DNA molecules into smaller fragments and how ligases reassemble them 2. perform simulations to demonstrate the use of restriction enzymes and ligases 3. explain, in general, how cells may be transformed by inserting new DNA sequences into their genomes

DNA and Biotechnology

Advancements● Genetic research in biotechnology (‘living technology’)

- advances in biological, agricultural, medical technology ● Recombinant DNA: fragment of DNA composed of

sequences originating from at least 2 different sources - move DNA sequences from one molecule to the otherEx) introduce gene encoding for insulin into bacterial cell DNA, they begin to produce insulin!

● Genetic Transformation: introduction and expression of foreign DNA in a living organism

DNA Sequencing● Before DNA is used for recombination, it must be sequenced ● Use Gel electrophoresis ● Sequence identified, isolated

- determines the exact order of bases ● Cut into many fragments of different lengths using restriction

enzymes ● 4 identical DNA samples labelled with different radioactive

markers (C, A, T, G) ● Loaded into 4 lanes of the gel ● Connect to electrical source: gel has positive end that attracts

fragments, negative end that repels them ● Move across the gel and markers show sequence. Must be

converted into SAMPLE DNA strand by complementary base pairing

Gel ElectrophoresisLaneA LaneG LaneC LaneT

Human Genome Project● Began October 1990, James Watson one of directors ● Human genome: 30 000 genes, 23 pairs of chromosomes

made up of estimated 3 billion pairs of nucleotides ● Used mapping techniques and DNA sequencing ● Pieces of DNA replicated and changed so that fragments,

each ending with one of the four nucleotides, can be detected with a laser

● Can then determine exact order of nucleotides in chain - computer compiles data to reconstruct original DNA sequence

● Completed in April 2003 Human Genome Project If you have time/are interested, watch a few of these: Ethical Concerns

Enzymes & Recombinant DNA● Recombinant DNA: ● Enzymes ● Restriction Endonucleases ● Methylases ● DNA Ligase ● Taq DNA Polymerase & Polymerase Chain Reaction

Restriction Endonucleases

● Restriction enzymes: act as scissors to cut DNA at specific base pair sequence

● Recognition sites: 4-8 base pairs long- palindromic sequence: both strands read the same in 5’- 3’direction

● Sticky ends: overhangs produced by exposed nucleotides

http://www.dnalc.org/resources/animations/restriction.html

http://education-portal.com/academy/lesson/how-ligase-is-used-to-engineer-recombinant-dna.html#lesson

http://education-portal.com/academy/lesson/restriction-enzymes-function-and-definition.html#lesson

Restriction Endonucleases● Not all produce sticky ends

ex) SmaI produces blunt ends (fully base paired) ● Sticky ends more useful: can be joined more easily

through complementary base pairing to other sticky ends

● Restriction enzyme used depends on where sequence needs to be cut- don’t pick one that will cut in middle of gene of interest

● Restriction enzymes named after bacteria it originates from

DNA Ligase● Joins pieces of fragment DNA from sources ● Allows geneticists to create segments of DNA with specific

nucleotide sequences they want ● If cut by same restriction enzymes, DNA ligase joins sticky ends

a) Complementary sticky ends produced by HindIIIb) Hydrogen bonds form between complementary bases. DNA ligase creates bonds between nucleotides in the DNA backbone.c) if fragments are not complementary, then hydrogen bonds will not form.

Transformation● Foreign DNA incorporated into the genome of a cell ● Vector: delivery system to move foreign DNA into cell

- chosen based on size and sequence of foreign fragment

● Goal: express the gene product(s) to change the traits of the organism that receives the foreign DNA

● Transgenic: organism with foreign DNA

Transformation of Bacteria● Most common organism transformed ● Transgenic bacteria used:

- study gene expression or function - maintain stock of particular DNA fragment- synthesize useful gene product

● Stages: first identify & isolate DNA fragment to be transferred

● Introduce into vector: plasmids - small, circular double-stranded DNA molecules - contain genes, introduce fragment into plasmid DNA

● Both plasmid and fragment cut with same restriction enzyme

● Mixed together, incubated with DNA ligase

Transformation of Bacteria● Bacterial cells treated

to open pores of cell membrane- take up plasmid

● Then makes many copies of recombinant plasmid - each includes copy of foreign DNA

Transgenics in our world:● We have made transgenic

bacteria that will produce hormones that we then collect for hormone therapy treatments (ex. Insulin)

● Bioremediation of oil spills, smokestacks, PCBs, water

● Plants that are resistant to herbicides, pests, or drought (canola, corn, wheat varieties)

● Plants with higher nutrient levels (Golden rice high in iron and VitA)

● Animals – goats that secrete a human proteins in milk, goats that secrete silk in milk, pigs that lack antigens so they are useful as organ donors...

● http://www.sciencechannel.com/video-topics/sci-fi-supernatural/kapow-superhero-science-spider-silk-gene-goats.htm

Diagnosing Genetic Disorders● Prenatal: ● Ultrasound to see visible defects ● Amniocentesis + karyotype to see chromosomal disorders ● Chorionic villi sampling + karyotype ● DNA probe to find a genetic marker

PCR (Polymerase Chain Reaction)● Use PCR to amplify the amount of

DNA so that you have enough to easily analyze it

● the two strands of the DNA double helix are physically separated at a high temperature in a process called DNA melting.

● the temperature is lowered and the two DNA strands become templates for a DNA polymerase (often Taq Polymerase) to selectively amplify the target DNA.

● The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification.

DNA Microarray Analysis● Gather mRNA from 2 different cells ● The mRNA present tells you which genes are being

expressed in a cell ● Can compare healthy and cancerous cells, or pinpoint

genes that are responsible for particular functions, or determine how environment impacts gene expression

● http://www.hhmi.org/biointeractive/how-analyze-dna-microarray-data

Red: gene expressed in cell 1 only Green: gene expressed in cell 2 only Yellow: gene expressed in both cells

Treating Genetic Disorders● Gene Therapy: ● Gene vector carries

foreign DNA to target cell

● Done in somatic cells (patient is now healthy, but germ line still does not have the corrected gene)

Benefits of Genetic Research● Medical advancement (understanding causes, effects,

and developing treatments. Possibility to predict side effects)

● Nutritional gains (higher yield, increased nutrient content...)

Ethical Issues● Is information public or private property? (resource

belonging to all, only belongs to individual, profit as motive to do more research)

● Patenting (agricultural products, ex. Monsanto and Round-up Ready Canola rules, genetic tests)

● Do we know all of the risks? (environment impacts, health effects, need time to do studies)

● Are we better to spend money on genetic research or directly helping people in need?

● Which genetic conditions will we treat with gene therapy?

● How much control should parents have over the genetic design of their children?