DNA Technology G/T Biology I

Uses of DNA Technology Purposely mutate genes to find out what they do (must mutate developing embryo) Purposely mutate genes to find out what they do (must mutate developing embryo) Clone genes (put human genes into bacteria - makes a lot of copies of a gene to study it, sequence it, or make large amounts of a protein) Clone genes (put human genes into bacteria - makes a lot of copies of a gene to study it, sequence it, or make large amounts of a protein) See if an embryo has a defective gene (use complementary DNA pieces to “tag” a gene) See if an embryo has a defective gene (use complementary DNA pieces to “tag” a gene) Make vaccines (make large amounts of proteins that trigger the immune response) Make vaccines (make large amounts of proteins that trigger the immune response) Gene Therapy (putting genes into somatic or germ cells) Gene Therapy (putting genes into somatic or germ cells)

Uses of DNA Technology Involving Gel Electrophoresis Identify recessive alleles Identify recessive alleles Diagnosis of disease Diagnosis of disease Study of relatedness of species Study of relatedness of species Crime Solving (DNA fingerprinting) Crime Solving (DNA fingerprinting) Paternity Testing Paternity Testing Figuring out the sequence of a gene Figuring out the sequence of a gene

Restriction Enzymes – What are they and why do you need them? They are enzymes that cut DNA at very specific sequences They are enzymes that cut DNA at very specific sequences We need them for any kind of recombinant DNA technology (genetic engineering) – splicing genes into different DNA We need them for any kind of recombinant DNA technology (genetic engineering) – splicing genes into different DNA Ex. Putting the human insulin gene into bacteria so that we can get bacteria that multiply quickly to make buckets of human insulin for diabetics Ex. Putting the human insulin gene into bacteria so that we can get bacteria that multiply quickly to make buckets of human insulin for diabetics We need them for any DNA technology involving gel electrophoresis We need them for any DNA technology involving gel electrophoresis

EnzymeOrganism from which derived Target sequence (cut at *) 5' -->3' Ava IAnabaena variabilisC* C/T C G A/G G Bam HIBacillus amyloliquefaciensG* G A T C C Bgl IIBacillus globigiiA* G A T C T Eco RIEscherichia coli RY 13G* A A T T C Eco RIIEscherichia coli R245* C C A/T G G Hae IIIHaemophilus aegyptiusG G * C C Hha IHaemophilus haemolyticusG C G * C Hind IIIHaemophilus inflenzae RdA* A G C T T Hpa IHaemophilus parainflenzaeG T T * A A C Kpn IKlebsiella pneumoniaeG G T A C * C Mbo IMoraxella bovis*G A T C Mbo IMoraxella bovis*G A T C Pst IProvidencia stuartiiC T G C A * G Sma ISerratia marcescensC C C * G G G SstIStreptomyces stanfordG A G C T * C Sal IStreptomyces albus GG * T C G A C Taq IThermophilus aquaticusT * C G A Xma IXanthamonas malvacearumC * C C G G G

What is Electrophoresis? o Electrophoresis is the separation of molecules (pieces of DNA) in a porous matrix based on electrical charge and size o The porous matrix is a gel o If you put an electrical charge through the gel material and put the DNA at the negative pole, it will migrate to the positive pole since DNA is negatively charged o If you cut the DNA into pieces and then run it thru the gel, the bigger pieces of DNA will move slower, the smaller pieces will move faster – therefore the pieces of DNA will separate

How do we use Electrophoresis to Diagnose a Disease or Identify a Recessive Allele Cut the DNA (if you have purified the gene) Cut the DNA (if you have purified the gene) Run gel (DNA runs toward the + pole) Run gel (DNA runs toward the + pole) Stain gel Stain gel CCG↓CGGTAGGAAC CCACGGTAGGAAC CCG↓CGGTAGGAAC CCACGGTAGGAAC ____ ____________

How do you use Electrophoresis for DNA Fingerprinting? Even though human DNA is 99.9% the same, 0.1% is 6 million differences in individual nucleotides. Even though human DNA is 99.9% the same, 0.1% is 6 million differences in individual nucleotides. Through research, we have identified regions of the DNA that is the most variable and cut those areas with restriction enzymes Through research, we have identified regions of the DNA that is the most variable and cut those areas with restriction enzymes This will make different sized fragments based on the enzymes cutting the DNA differently This will make different sized fragments based on the enzymes cutting the DNA differently If we run them on a gel, we will see different patterns If we run them on a gel, we will see different patterns The same person always has the same pattern because the DNA doesn’t change The same person always has the same pattern because the DNA doesn’t change Different people may have the same pattern if they have the same mutations in the areas of the DNA analyzed Different people may have the same pattern if they have the same mutations in the areas of the DNA analyzed

This is an example of a real DNA fingerprint from a real case

How to Use Electrophoresis for Paternity Testing Cut the DNA up with restriction enzymes and run it on a gel Cut the DNA up with restriction enzymes and run it on a gel Every allele the child has, must come from the mother or father Every allele the child has, must come from the mother or father If you know who the mother is, any band that didn’t come from her, must be from the father If you know who the mother is, any band that didn’t come from her, must be from the father

PCR – polymerase chain reaction Make millions of copies of a single piece of DNA (like DNA replication in a test tube) Make millions of copies of a single piece of DNA (like DNA replication in a test tube) DNA can be old and in very small quantities DNA can be old and in very small quantities Can use for crime detection if only have 1 cell or a small sample Can use for crime detection if only have 1 cell or a small sample Basically, throw in DNA into a test tube with nucleotides, DNA polymerase Basically, throw in DNA into a test tube with nucleotides, DNA polymerase Heat it up to separate the DNA and cool it down for it base pair back together Heat it up to separate the DNA and cool it down for it base pair back together The DNA polymerase is special – isolated from bacteria that live at 160 degree water The DNA polymerase is special – isolated from bacteria that live at 160 degree water

A. Double strand DNA B. Denature 96º 50º C. Anneal primers 50º D. Polymerase binds 72º Taq

72º Taq E. Copy strands F. Denature 96º Taq