DNA Technology

Biotechnology The use or alteration of cells or biological molecules for specific applications Transgenics Transgenic “changed genes” Recombinant DNA DNA from different species mixed together Natural or man-made Whole organisms or cells Possible because the genetic code is universal All life uses the same genetic code (A,T,G,C)

Amplifying DNA Need many copies for various DNA tests from a small initial sample Two techniques Polymerase chain reaction (PCR) Recombinant DNA technology

PCR Done on molecules Based on DNA replication Rapidly replicates a selected sequence of DNA in a test tube Used to: Establish blood relationships Identify remains Convict or exonerate suspects Look at pathogens Identify genes Very sensitive but easily contaminated

Review of DNA Replication Parental molecule of DNA Both parental strands serve as templates Two identical daughter molecules of DNA T Nucleotides CG A GC A A T T A A A A C C C T T G G A C A A A A A A CC C CG T G T T T T G T T T G G

Requirements for PCR Know parts of the DNA sequence to be amplified 2 DNA Primers Short, lab-made single-stranded DNA One complementary for each strand in the DNA segment to be amplified Lots of nucleotides Taq DNA polymerase From Thermus aquaticus (lives in hotsprings) A thermal cycler

PCR

Recombinant DNA (cloning) Works in cells Adds genes from one type of organism to the genome of another Requires: Restriction enzyme Vector Donor DNA Host bacteria

Restriction Enzymes Figure 19.3a

Cloning Vectors Carries DNA from the cells of one species into cells of another Any piece of DNA into which another can be inserted

Types of Vectors Which vector used depends on size of the gene to be inserted Plasmid One type of cloning vector Small circle of double-stranded DNA Occurs naturally in some bacteria and yeasts

Creating Recombinant DNA Molecules Figure 19.3a

Figure 19.3b

Recombinant DNA Figure 19.5

Selecting Recombinant DNA Molecules 3 types of cells Cells that lack plasmids Cells that contain plasmids that do not contain a foreign gene Cells that contain plasmids that have the foreign gene – WANT THESE Plasmids can contain a gene for an enzyme that catalyzes a reaction that makes a blue color If a foreign gene inserts in the middle of this “blue” gene, the bacteria containing the plasma will not produce the blue color Plasmids can contain a gene for antibiotic resistance When the antibiotic is applied to the bacterial cells, only those with the plasmid will survive

Applications of Recombinant DNA Drugs (e.g. insulin) Pure, human versions e.g. “humulin” Transgenic plants a.k.a. genetically modified (GM) plants Transgenic animals e.g. 3xTg-AD mouse

Transgenic Plants

Transgenic Animals

Isolating a Gene Make a DNA probe Strand of DNA complementary to the one of interest Attached to a radioactive or fluorescent molecule

Complementary (cDNA) Library Represents only protein-encoding genes Made from mRNAs Represents the proteins being made in a particular cell

Making a cDNA library Extract mRNAs from cells Use reverse transcriptase to make DNA from RNA Use DNA polymerase to make double-stranded DNA