GH1

gene that codes for HGH

defect in it = pituitary dwarfism

prions

infectiuous proteins

reverse transcriptase

-allows information to flow from RNA to DNA

-catalyzes the synthesis of DNA from RNA template

complementary DNA

(cDNA)

-DNA that is produced via R.T. from RNA

2 ways to get double helix from R.T.

R.T. will go again synthesizing strand to produce double strand 

OR

DNA polymerase will crease double strand

DNA cloning

efforts to produce many copies of a gene

(insert gene into plasmid

insert modified plasmid into bacterial cell,

replicate many bacterial cells)

plasmid

small cirular DNA molecule

(bacteria)

Cloning vector

when plasmid is used to make copies of foreign DNA sequence

Restriction endonuclease

bacterial enzyme that cuts DNA molecules at SPECIFIC base sequences

(cut DNA from invading viruses)

4 steps to inserting genes into plasmid

(1) identify a palindromic recognition site

(2) add restriction endonucleaste

-makes staggered cuts

(3) sticky ends result

-recognition sites now have sticky ends capable of H bonds

(4) Insert Gene into Plasmid

-stick ends bind to complementary base pairs

-DNA ligase catalyzes formation of phosphodiester bonds ("sealing" the gene)

sticky ends

short single stranded ends of DNA molecule

-cut by restriction endonuclease

form H bonds with other sticky ends at complementary bases

DNA ligase

joins pieces of DNA by catalyzing the formation of a phosphodiester bont btwn the pieces

Recombinant DNA technology

-ability to create various combinations of DNA sequences by cutting specific sequences and pasting them into new location

transformation

cells that take up DNA from environment and incorporate them into their genomes

DNA library

collection of DNA sequences, each of which is inserted into a vector DNA library

cDNA library

if the sequences are cDNAs from a particuar cell type/tissue

genomic library

sequences are fragments of DNA that collectively represent the entire genome of an organism

how to produce a cDNA library

(1) isolate mRNAs

(2) synthesis cDNA (reverse transcriptase)

(3) make cDNA doulbe stranded (by R.T. or DNA poly)

(4) make recombinant plasmid (insert each double stranded DNA into different plasma)

(5) transfomation (intoduce recombinant plasmids into E coli cells by making cells permeable to DNA)

Probe

a marked molecule that binds to the molecule that is being looked for

-single stranded fragment that will bind to complementary bases of strand being analyzed

6 steps in screening a cDNA library

(1) grow Transformed E.Coli

-cells containing plasmids can grow on plates-each colony = diff cDNA

(2) Lay a filter on each plate then remove (cells stick)

(3) treat bacteria w/ chemicals to make single stranded DNA

(4) Probe Filters

(5) Find probe

(6) identify colony

Polymerase Chain Raction 

PCR

in virtro DNA synthesis rxn in which a specific seciton of DNA is replicated over and over by DNA poly to amplicfy the # of copies of that sequence

PCR steps

(1) start w/a solution containing a template DNA, syntehsized primers, taq polyerase, dNTPs and buffers

(2) DENATUATION -heating separat strands in double helix

(3) PRIMER ANNEALING - at cooler temps primers bind to template DNA by complementary base paring

(4) EXTENSION -during incubation Taq polymerase uses dNTPS to synthesize complementary DNA strand, starting at primer

(5) repeat 2-4

(6) repeat again

Temperatures of Denatuation

annealing and extension

denatuation - 95 C

annealing - 50-60 C

extension- 72 C

Taq polymerase

-DNA poly found in bacteria

-used in the PCR rxn bc heat stable

-optimal temp = 72 C

Dideoxy Sequencing

variation on the basic in vitro DNA synthesis rxn

Steps in dideoxy sequencing

(1) Incubate RXN mixture (dNTPs-extend DNA strand; ddNTPS-terminate synthesis; template DNA; primer for target sequence; DNA poly)

(2) DNA sysnthesis occurs

(3) Collect DNA strand produced (fragements of newly syntehsized DNA have distinc labels on them)

(4) Separate Fragments (via electrophoesis)

(5) read output

difference btwn ddNTPs and dNTPs

ddNTPs lack a hydroxyl group on the 3' carbon

(thats why they cause termination

-phosphodiest bonds cand form)

uses of genetic engineering

-study structure and function of a specific gene

-study where when and how its expression occurs

-find genes associated with dieseas

-potential to use gene theraphy to correct defect genes

-improve agriculatual crops