Chapter 19

Front 1

Analyzing and engineering genes

Back 1

Birth of genetic engineering, recombinant DNA technology
discovery of bacterial enzymes that cut DNA at specific sequences and paste DNA back together

Front 2

Recombinant DNA techniques to make proteins

Back 2

Pituitary Dwarfism
Pituitary gland produces growth hormone GH1 141 amino acids
Type 1- defect in GH1 gene, little or no hormone produced, autosomal recessive trait, short in stature, 2-10 years late in reaching puberty

Front 3

Treating Pituitary Dwarfism

Back 3

Injectng growth hormone from other species didnt work, human growth hormone from cadavers did work but very scarce and expensive. Children treated developed Creutzfelt Jakob disease- prions=misfolded proteins.
1984 use of hormone from cadavers was banned

Front 4

Another way to treat

Back 4

Insert copies of GH1 gene into E Coli to grow large quantities of the protein. Isolate gene, make copies, insert into ecoli

Front 5

Isolate the gene

Back 5

GH1 expressed in pituitary gland, isolate mRNA from pituitary gland. use reverse Transcriptase to amke cDNA (complementary DNA) copy of the mRNA.
Add primer and DNA polymerase to make double stranded cDNA

Front 6

Cloning

Back 6

Make many copies of the gene. Insert gene into small circular DNA molecule called plasmid--found in bacterial cells, distinct from chromosome and replicate independently, carry antibiotic resistance, cloning vector carry foreign DNA and make copies

Front 7

Restriction endonucleases

Back 7

Bacterial enzymes that cut DNA at specific sequences, often cut at palindrome sequence. Enzymes create a "sticky end", single stranded bases on one fragment are complementary to the single stranded bases on another fragment

Front 8

Transformation

Back 8

Introduction of recombinant plasmids into bacterial cells
Increase permeability of cells by chemical treatment or electric shock
Plate bacterial cells on media in a thin layer so each cell can grow individually

Front 9

Making human growth hormone

Back 9

Inserted GH1 gene into plasmid, plasmid contains promoter, cDNA doesn't ahve introns, no splicing needed.
Inserted plasmid into E coli, GH1 is produced in cells, isolated and purified
Safe and reliable source of GH1, successful treatment
ethical issues in athletic performance and appearance

Front 10

Polymerase Chain Reaction

Back 10

Another way to copy DNA
Specific section of DNA is replicated over to make copies
Need to have info about DNA sequence to do PCR, and need to synthesize primers for each side of the gene
Tag polymerase = DNA polymerase from Thermus aquaticus discovered in hot spring in yellow stone, heat stable and works at high temp

Front 11

Ways to use PCR

Back 11

Study ancient fossil DNA- sequence & compare DNA
Forensics DNA from crimescene
Genetic counseling, check for disease in baby
Evolutionary studies how organisms relate
Kary Mullis

Front 12

Gel electrophoresis

Back 12

Move toward positive side cause DNA is negative because of the phosphate group. Separate nucleic acids and look at size

Front 13

DNA Sequencing

Back 13

Understand difference in normal and mutant genes. Infer protein sequence from DNA sequence, to infer how closely related species are

Front 14

Dideoxy sequencing

Back 14

Frederick Sanger- Dideoxynucleotide (ddNTP) will determine DNA synthesis cause no 3' hydroxyl group for next dNTP
Start w/ a labeled primer, each strand labeled and given different length
Use all 4 ddNTPs=ddATP, ddCTP, ddGTP, ddTTp

Front 15

Mapping Plasmid DNA with Restriction Enzymes

Back 15

Clone the gene of interest into plasmid, cut with restriction enzymes, separate fragments by electrophoresis, map locations of restriction enzyme sites and distances between them
bp=1 base pair, 1000 bp=1kilobase

Front 16

Mapping DNA fragments with restriction enzymes

Back 16

Cut plasmid with each enzyme separately, then with both enzymes together
Analyze sizes of fragments by electrophoresis
Put fragments together to build a map of plasmid, sizes must add up correctly