intro to molecular genetics restriction enzymes mapping cloning pcr sequencing genetic engineering

INTRO TO MOLECULAR GENETICS

•Restriction enzymes

•Mapping

•Cloning

•PCR

•Sequencing

•Genetic engineering

A restriction enzyme cuts DNA at a specific sequence

(Bacteria are safe because their DNA is methylated (with a CH3 group) at these sites.)

http://www.mun.ca/biology/scarr/RFLP_test_for_recessive_trait.html

RFLPs were an early form of genetic marker

Click here to see a map of Chromosome 6 in humans:

http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?taxid=9606&chr=6

Various kinds of information were used to make these maps.

Chromosome 6 contains about 1500 genes. You can see more of them if you zoom in on the right-most map.

Molecular Cloning -- make many copies of certain strands of DNA

If use messanger RNA, as here,will clone DNA that is expressedin a certain tissue.

If clone into “expression vector”can make gene product.

Can also clone “genomic DNA”, which will include non-coding regions.

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Polymerase Chain Reaction (PCR)

Efficient way to make many copies of a piece of DNA.Has replaced cloning for some applications.You will need: •Double stranded DNA (the template)•Primers (two ~ 20 bp single-stranded oligonucleotides that are

complementary to the template, spanning the region of interest.)•DNA polymerase, preferably from a hot-spring bacterium•dNTPs (dioxynucleotide triphosphates A, C, G, and T)•Buffer to run the reaction in

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PCR repeats 3 steps:

1. Denaturation

2. Primer annealing

3. Extension

At each repetition, the numberof copies of DNA doubles.

It’s fun and easy!

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Normal (deoxy) dNTP(extends strand)

Di-deoxy nucleotide ddNTP(terminates extension)

DNA sequencing

Requires:A mix of dNTPs and

ddNTPsPolymeraseA labeled primer

Generates:

A mix of fragments.The length of each fragment indicates which ddNTP was added, andtherefore the base at thatposition.

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We use

automated

sequencing

here at Colby.

What do we do with this new information?

Genetic testing

Improved understanding of phenotype & treatments

Information on relatedness of populations and species

Genetic engineering?

Retroviruses can carry the desired DNA into human cells.These viruses carry reverse transcriptase, which uses RNA as a template to make DNA.Sometimes, this fragment will be incorporated into the host (human) genome.

Problems:Viruses are scary and germy!Immune system response can be serious.The fragment could happen to land in the middle of a functional gene.

More feasible: implant stem cells that have undergone genetic engineering.

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both viral and human sequences.

genes enter host cell. stranded DNA

version of introduced genes.

Genetic engineering is widespread in agriculture.

A descriptive animation of cell culture from Agrobacterium transformation:

http://www.agriculture.purdue.edu/agbiotech/images/leafdisk1.html

A descriptive animation of the gene gun:

http://www.agriculture.purdue.edu/agbiotech/images/Genegun1.html

Agrobacterium infects many plants.

This bacterium contains a plasmid, an extra-chromosomal piece of DNA.

A section of the plasmid is incorporated into the chromosomes of the cells (virus-like), causing them to grow and divide rapidly.

Humans can replace these “tumor” genes with genes of our choice.

Examples of genetically engineered crops:(the FDA has completed consultations on these crops* )

Soybean -- herbicide resistanceCorn -- resistance to herbivory by insects; herbicide resistanceSugar beet -- herbicide resistance (the final product contains no DNA or protein)

Alfalfa -- herbicide resistanceWheat -- herbicide resistanceRice -- herbicide resistanceCanteloupe -- delayed ripening due to reduced ethyleneTomato -- resistance to herbivory by insects; delayed ripeningPotato -- resistance to virus and beetlesSquash -- resistance to virusesPapaya -- resistance to viruses

*http://vm.cfsan.fda.gov/%7Elrd/biocon.htmlInteresting Purdue website: http://www.agriculture.purdue.edu/agbiotech/onthetable.html