DNA TechnologyDNA Technology

G/T Biology IG/T Biology I

Uses of DNA TechnologyUses of DNA Technology Purposely mutate genes to find out what they do Purposely mutate genes to find out what they do

(must mutate developing embryo)(must mutate developing embryo) Clone genes (put human genes into bacteria - Clone genes (put human genes into bacteria -

makes a lot of copies of a gene to study it, makes a lot of copies of a gene to study it, sequence it, or make large amounts of a protein)sequence it, or make large amounts of a protein)

See if an embryo has a defective gene (use See if an embryo has a defective gene (use complementary DNA pieces to “tag” a gene)complementary DNA pieces to “tag” a gene)

Make vaccines (make large amounts of Make vaccines (make large amounts of proteins that trigger the immune response)proteins that trigger the immune response)

Gene Therapy (putting genes into somatic or Gene Therapy (putting genes into somatic or germ cells)germ cells)

Uses of DNA Technology Involving Uses of DNA Technology Involving Gel ElectrophoresisGel Electrophoresis

Identify recessive allelesIdentify recessive allelesDiagnosis of diseaseDiagnosis of diseaseStudy of relatedness of speciesStudy of relatedness of speciesCrime Solving (DNA fingerprinting)Crime Solving (DNA fingerprinting)Paternity TestingPaternity TestingFiguring out the sequence of a geneFiguring out the sequence of a gene

Restriction Enzymes – What are Restriction Enzymes – What are they and why do you need them?they and why do you need them?

They are enzymes that cut DNA at very specific They are enzymes that cut DNA at very specific sequencessequences

We need them for any kind of recombinant DNA We need them for any kind of recombinant DNA technology (genetic engineering) – splicing technology (genetic engineering) – splicing genes into different DNAgenes into different DNA Ex. Putting the human insulin gene into bacteria so Ex. Putting the human insulin gene into bacteria so

that we can get bacteria that multiply quickly to make that we can get bacteria that multiply quickly to make buckets of human insulin for diabeticsbuckets of human insulin for diabetics

We need them for any DNA technology involving We need them for any DNA technology involving gel electrophoresisgel electrophoresis

Enzyme Organism from which derived Target sequence(cut at *)5' -->3'

Ava I Anabaena variabilis C* C/T C G A/G G

Bam HI Bacillus amyloliquefaciens G* G A T C C

Bgl II Bacillus globigii A* G A T C T

Eco RI Escherichia coli RY 13 G* A A T T C

Eco RII Escherichia coli R245 * C C A/T G G

Hae III Haemophilus aegyptius G G * C C

Hha I Haemophilus haemolyticus G C G * C

Hind III Haemophilus inflenzae Rd A* A G C T T

Hpa I Haemophilus parainflenzae G T T * A A C

Kpn I Klebsiella pneumoniae G G T A C * C

Mbo I Moraxella bovis *G A T C

Mbo I Moraxella bovis *G A T C

Pst I Providencia stuartii C T G C A * G

Sma I Serratia marcescens C C C * G G G

SstI Streptomyces stanford G A G C T * C

Sal I Streptomyces albus G G * T C G A C

Taq I Thermophilus aquaticus T * C G A

Xma I Xanthamonas malvacearum C * C C G G G

What is Electrophoresis?What is Electrophoresis?o Electrophoresis is the separation of molecules Electrophoresis is the separation of molecules

(pieces of DNA) in a porous matrix based on (pieces of DNA) in a porous matrix based on electrical charge and sizeelectrical charge and size

o The porous matrix is a gelThe porous matrix is a gelo If you put an electrical charge through the gel If you put an electrical charge through the gel

material and put the DNA at the negative pole, it material and put the DNA at the negative pole, it will migrate to the positive pole since DNA is will migrate to the positive pole since DNA is negatively chargednegatively charged

o If you cut the DNA into pieces and then run it If you cut the DNA into pieces and then run it thru the gel, the bigger pieces of DNA will move thru the gel, the bigger pieces of DNA will move slower, the smaller pieces will move faster – slower, the smaller pieces will move faster – therefore the pieces of DNA will separatetherefore the pieces of DNA will separate

How do we use Electrophoresis to Diagnose a How do we use Electrophoresis to Diagnose a Disease or Identify a Recessive AlleleDisease 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 gelStain gel

CCGCCG↓CGGTAGGAAC CCACGGTAGGAAC↓CGGTAGGAAC CCACGGTAGGAAC

________

________

________

How do you use Electrophoresis for How do you use Electrophoresis for DNA Fingerprinting?DNA Fingerprinting?

Even though human DNA is 99.9% the same, 0.1% is 6 Even though human DNA is 99.9% the same, 0.1% is 6 million differences in individual nucleotides. million differences in individual nucleotides.

Through research, we have identified regions of the DNA Through research, we have identified regions of the DNA that is the most variable and cut those areas with that is the most variable and cut those areas with restriction enzymesrestriction enzymes

This will make different sized fragments based on the This will make different sized fragments based on the enzymes cutting the DNA differentlyenzymes cutting the DNA differently

If we run them on a gel, we will see different patternsIf we run them on a gel, we will see different patterns The same person always has the same pattern because The same person always has the same pattern because

the DNA doesn’t changethe DNA doesn’t change Different people may have the same pattern if they have Different people may have the same pattern if they have

the same mutations in the areas of the DNA analyzedthe 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 How to Use Electrophoresis for Paternity TestingPaternity Testing

Cut the DNA up with Cut the DNA up with restriction enzymes and restriction enzymes and run it on a gelrun it on a gel

Every allele the child has, Every allele the child has, must come from the mother must come from the mother or fatheror father

If you know who the If you know who the mother is, any band that mother is, any band that didn’t come from her, must didn’t come from her, must be from the fatherbe from the father

PCR – polymerase chain reactionPCR – polymerase chain reaction Make millions of copies of a single piece of DNA Make millions of copies of a single piece of DNA

(like DNA replication in a test tube) (like DNA replication in a test tube) DNA can be old and in very small quantitiesDNA can be old and in very small quantities Can use for crime detection if only have 1 cell or Can use for crime detection if only have 1 cell or

a small samplea small sample Basically, throw in DNA into a test tube with Basically, throw in DNA into a test tube with

nucleotides, DNA polymerasenucleotides, DNA polymerase Heat it up to separate the DNA and cool it down Heat it up to separate the DNA and cool it down

for it base pair back togetherfor it base pair back together The DNA polymerase is special – isolated from The DNA polymerase is special – isolated from

bacteria that live at 160 degree waterbacteria that live at 160 degree water

A. Double strand DNA

B. Denature96º

50º

C. Anneal primers

50º

D. Polymerase binds

72ºTaq

Taq

72ºTaq

Taq

E. Copy strands

1

2

3

4

F. Denature

96º

Taq

Taq