G ENETIC T ECHNOLOGY

1) G ENETIC C LONING 1) G ENETIC C LONING O VERVIEW 1. Remove bacterial plasmid with restriction enzymes 2. Add in gene of interest (plasmid is now recombinant DNA molecule) 3. Put back into bacteria 4. Many reproductive cycles later = amplification of gene & protein it makes

R ESTRICTION E NZYMES Cut up foreign DNA Very specific Recognize short nucleotide sequences  cut at specific points within sequence Bacteria’s own DNA is methylated to protect itself What could we use this for????

R ESTRICTION E NZYMES Enzyme finds specific recognition sequence (restriction site) Same sequence found on both strands, running antiparallel Enzyme cuts phosphodiester bonds of strands These restriction fragments are double stranded with single stranded ends (“sticky ends”)

R ESTRICTION E NZYMES Single strands will hydrogen bond with other complementary “sticky ends” Bonds made permanent with DNA ligase Now we have recombinant DNA

F ROM R ESTRICTION E NZYMES TO P LASMID M APS PM show how different REs act upon a plasmid Pictorial representation of the different lengths of pieces remaining after the REs worked

F ROM R ESTRICTION E NZYMES TO P LASMID M APS Procedure Think of plasmid as clock – from 12 to 12 = total # base pairs Approximate location of cut based on base pair fragment length Use logic to solve Double check based on data

G ENETIC R ECOMBINATION Occurs with help of plasmids (cloning vector) DNA molecule that can carry foreign DNA into a cell & replicate there

2) P OLYMERASE C HAIN R EACTION 2) P OLYMERASE C HAIN R EACTION (PCR) Can quickly amplify specific DNA without using cells DNA of interest incubated with DNA polymerase, nucleotides, & ss primer DNA for synthesis DNA heated  strands separate Cool  primers bond DNA polymerase adds to 3’ end of each primer Repeat

3A) DNA A NALYSIS (GE) Sequence of entire genome  genomics Begins with gel electorphoresis Sorts DNA based on size & charge Can combine with specific probes to label particular DNA bands

3B) DNA A NALYSIS (SB) Can also use Southern blottingSouthern blotting Helps to detect restriction fragment length polymorphisms (RFLP)restriction fragment length polymorphisms Differences in DNA sequences on homologous chromosomes Can result in different patterns of restriction fragment lengths Genetic marker for making linkage maps Led to Human Genome Project

4) G ENOME A NALYSIS Scan sequence for start & stop codes, RNA splicing sites, known genes Found 30,000-40,000 genes Can figure out new genes by comparing to old genes with similar sequence

4) G ENOME A NALYSIS DNA microarrayDNA microarray: ssDNA fragments fixed to slide that are then labeled with fluorescent cDNA Compare genes of species  attempt to uncover gene functiongene function

5) G ENOME A NALYSIS - G ENE F UNCTION To determine, turn gene off – see what happens To turn off: RNA interference (RNAi)RNA interferenceRNAi Synthetic ds RNA matches gene sequence – binds to mRNA Triggers breakdown of mRNA  no protein made Remove to turn on again

T HE F UTURE Proteomics: study of full protein sets Study of variations among the species Form of single nucleotide polymorphisms (SNPs) Single base-pair variations One per 1000 bp

DNA T ECHNOLOGY A PPLICATIONS Disease Diagnosis Use PCR & labeled nucleic acid probes to detect pathogens (ex: HIV) Identification of harmful alleles before birth

DNA T ECHNOLOGY A PPLICATIONS Human Gene Therapy Alteration of genes Replace defective gene with normal one  put into cells that keep dividing Appears to be temporary Raises ethical questions

DNA T ECHNOLOGY A PPLICATIONS Pharmaceutical Products Use vector DNA to create human insulin, HGH, TPA, etc Recombinant DNA to make vaccine without using actual pathogen

DNA T ECHNOLOGY A PPLICATIONS Forensics Microsatellite DNA highly variable between individuals Called simple tandem repeats (STR) Environmental Genetically engineered microbes to degrade toxic waste

DNA T ECHNOLOGY A PPLICATIONS Agriculture Transgenic organisms: carry genes from another species Makes “super” species Remove egg  fertilize in vitro  inject desired DNA into egg nuclei  cell will grow & express gene  egg put into surrogate

DNA T ECHNOLOGY A PPLICATIONS Plants Vector is recombinant Ti plasmid – inserts into plant genome  cell grows into complete plant Can increase nutritional value