GENETIC ENGINEERINGCHAPTER 20

• Genetic engineering is the ability of humans to modify and manipulate DNA for:– Identification of genetic disorders– Gene therapy– Crop and food production– Tailoring medicines for the cancer– Creating recombinant medicines, vaccines– Forensics– Analyzing evolutionary relationships

• In order to do these, scientists need the genes to study and sequence

I. DNA Cloning

• Involves inserting DNA into a vector and cloning it into a cell for production of multiple copies

A. Vectors– DNA molecules that will carry foreign DNA into

cells: plasmids, BACs, YACs

• Plasmid: small circular DNA found in bacteria.

• • Antibiotic resistant gene allows selection of bacteria

that took up plasmid• Multiple cloning site allows insertion of foreign DNA

• Bacterial artificial chromosome (BAC): large plasmid that can carry large DNA fragments in bacterial cells

• Yeast artificial chromosome (YAC): derived from yeast DNA and used to clone really large DNA fragments into eukaryotic cells

B. restriction enzymes• found naturally in bacteria to protect them from

invading viruses• molecular scissors that cut DNA at specific

nucleotide sequences• enzyme binds to DNA at that sequences and cuts

between the sugar and phosphate on both strands

– cuts can leave blunt or sticky ends

Constructing recombinant DNA:

II. How to clone a gene using a plasmid vector

A. Basic procedure– Cut the DNA of interest and vector with the same

restriction enzyme (genomic or cDNA)– Fragments are mixed and ligase seals fragments

together– Vector DNA will incorporate the fragments of source

DNA in them creating recombinant plasmid– Some vectors will NOT incorporate foreign DNA. They

are nonrecombinant– Cells are transformed by mixing vector with cells– Recombinant cells are selected for using agar plates

with antibiotics

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B. DNA libraries• Contain fragmented DNA

from a particular species stored in a vector ready for cloning– Genomic libraries: made

from genomic DNA – cDNA libraries: made from

a species’ mRNA using reverse transcriptase

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Constructing a cDNA library:

C. Screening for the gene of interest

• Use a radioactive probe that will only recognize gene of interest– Transfer some of the bacteria to a piece of filter

paper forming a replica– Incubate filter with radioactive piece of DNA

complementary to gene of interest– Make an X-ray film.– Use the X-ray film to isolate colony with gene of

interest

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III. DNA technology A. PCR• Makes many copies of a

DNA sample• Uses a three step cycle:• Heating to about 95o

C to separate strands• Cooling to anneal

primers• Replication using ___

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B. Electrophoresis and Blotting1. Electrophoresis• Uses a gel as a

molecular sieve to separate nucleic acids or proteins based on size

• A current is applied to gel that causes the charged molecules to move thru the gel forming bands based on size

• Bands are visualized with some type of dye

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2. Blotting–After electrophoresis, bands are transferred

to a filter paper for analysis of a particular fragment (a gene fragment, mRNA, or protein)–Filter paper is incubated with a “probe” to

the particular fragment of interest –Southern blot analyzes DNA –Northern blot analyzes mRNA–Western blot analyzes proteins– http

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Northern blot

3. DNA sequencing

• Small stretches of DNA are sequenced using dideoxy chain termination method

• Uses dideoxynucleotides (ddNTP) mixed with normal ones

• Each type of ddNTP has a different fluorescent tag

• DNA sequence is read

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Automated sequencing done now:

Manual sequencing done in 1980s and 1990s

IV. The human genome is polymorphic

• Genetically, humans are 99% identical but:– Alleles have small nucleotide differences – Different individuals have different numbers of

short tandem repeats: a sequence of 2-5 nucleotides repeated over

– This is restriction fragment length polymorphism

SNPs:

Short Tandem Repeats:

A. Creating genetic profiles using STR analysis

• There are many regions on chromosomes where STRs exist.

• The number of tandem repeats in each region varies from individual

• By using PCR on the STR’s of an individual, you can create a genetic profile

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Cloning an organism: