DNA TechnologyDNA Technology

A.P. BiologyA.P. Biology

Biotech Intro

Glowing Fish Glowing Mouse Mistake

What’s Next – Google Brain?What’s Next – Google Brain?

Uses of DNA TechnologyUses of DNA TechnologyFind out what genes do (mutate or knock Find out what genes do (mutate or knock

out a developmental gene and see what out a developmental gene and see what happens)happens)

Make large amounts of a proteinMake large amounts of a protein In-situ hybridization – can tell if an embryo In-situ hybridization – can tell if an embryo

has a defective genehas a defective geneDiagnosis, treatment, prevention of Diagnosis, treatment, prevention of

diseasediseaseStudy of relatedness of speciesStudy of relatedness of speciesCrime SolvingCrime Solving

DNA Technology Uses ContinuedDNA Technology Uses Continued Study gene expressionStudy gene expression Study growth and differentiationStudy growth and differentiation Identify recessive allelesIdentify recessive alleles Vaccines (make large amounts of proteins that Vaccines (make large amounts of proteins that

trigger the immune responsetrigger the immune response Designer Drugs (anti-fat drugs)Designer Drugs (anti-fat drugs) RNAi (blocks translation – find out what a gene RNAi (blocks translation – find out what a gene

does)does) Gene Therapy (put genes in somatic or germ Gene Therapy (put genes in somatic or germ

cells)cells) Problems:Problems: How do you introduce it?How do you introduce it? How do you control gene expressionHow do you control gene expression How do you get the gene product where it’s neededHow do you get the gene product where it’s needed May alter other cell functionsMay alter other cell functions Eugenics are a worry (controlling the genetic make-up)Eugenics are a worry (controlling the genetic make-up)

Technology #1 – Gene CloningTechnology #1 – Gene Cloning

Gene CloningGene CloningMaking multiple copies of a gene by putting it in a Making multiple copies of a gene by putting it in a cell that will replicate it and pass it on to offspringcell that will replicate it and pass it on to offspring

Although you can make lots of copies of a Although you can make lots of copies of a gene in a tt, it will actually produce the gene in a tt, it will actually produce the

protein if in a cellprotein if in a cell

To clone a gene you will need:To clone a gene you will need:restriction enzymes, a vector, and a host cellrestriction enzymes, a vector, and a host cell

Restriction EnzymesRestriction Enzymesa.a. naturally used by bacteria to digest foreign naturally used by bacteria to digest foreign

DNADNA

b.b. Bacteria methylates its DNA (A&C) to Bacteria methylates its DNA (A&C) to protect itself since methylation prevents protect itself since methylation prevents DNA digestionDNA digestion

c.c. Can buy and use to cut and insert foreign Can buy and use to cut and insert foreign DNADNA

d.d. Cut at palindromic sequences – same Cut at palindromic sequences – same forward and back (same 5’-3’ or vice versa)forward and back (same 5’-3’ or vice versa)

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

Cloning Vectors Cloning Vectors Something to carry gene so it gets copiedSomething to carry gene so it gets copied

a. a. PhagesPhages (as the phage replicates inside the bacteria so (as the phage replicates inside the bacteria so does the added gene and it spreads to other bacteria) does the added gene and it spreads to other bacteria) – – holds up to 25kb of DNAholds up to 25kb of DNA

b. b. PlasmidsPlasmids – as bacteria reproduce – – as bacteria reproduce – clones clones gene within the colony – gene within the colony – holds up to 12 kbholds up to 12 kb

c. c. RetrovirusesRetroviruses – have advantage that they can – have advantage that they can incorporate into the host chromosome in animal cells – incorporate into the host chromosome in animal cells – Can hold 8-10kbCan hold 8-10kb

d.d. Yeast artificial chromosomes Yeast artificial chromosomes (YAC’s) – a “fake” (YAC’s) – a “fake” chromosome containing foreign DNA with the ability chromosome containing foreign DNA with the ability to replicate and undergo mitosis – to replicate and undergo mitosis – up to 3000kbup to 3000kb

e. e. Bacterial Artificial ChromosomesBacterial Artificial Chromosomes (BAC’s) – a fake (BAC’s) – a fake bacterial chromosome – bacterial chromosome – can hold 100-300 kbcan hold 100-300 kb

Example of PlasmidExample of Plasmid

Advantages of Plasmids as VectorsAdvantages of Plasmids as Vectors

Replicate quicklyReplicate quicklyHas an origin of replication so is copied Has an origin of replication so is copied

and passes to daughter cellsand passes to daughter cellsDoesn’t need to enter genome to be Doesn’t need to enter genome to be

copiedcopiedEasy to put DNA into plasmids Easy to put DNA into plasmids Easy to put plasmids into bacteriaEasy to put plasmids into bacteriaCan incorporate selection factors to make Can incorporate selection factors to make

it easy to find bacteria containing gene of it easy to find bacteria containing gene of interestinterest

HostHostCan use animal cell, plant cell, yeast cell, or bacteria. Bacteria are the

easiest – can get DNA in to the cell easier and they replicate faster

a. Difficult to put DNA into eukaryotic cells (can a. Difficult to put DNA into eukaryotic cells (can electroporate, inject the DNA, or attach the DNA to electroporate, inject the DNA, or attach the DNA to metal beads and shoot through membrane with a metal beads and shoot through membrane with a gun) – usually use retroviruses!gun) – usually use retroviruses!

b. Although bacteria are easy to use, you can’t b. Although bacteria are easy to use, you can’t always use them because they don’t have always use them because they don’t have

mechanisms to cut out introns and don’t do post-mechanisms to cut out introns and don’t do post-translational modifications – if it needs these translational modifications – if it needs these modifications to be functional, must use a eukaryotic modifications to be functional, must use a eukaryotic cellcell

Making Recombinant DNA: Making Recombinant DNA: How do you clone a gene and how do find How do you clone a gene and how do find

the cells that have your gene in them?the cells that have your gene in them?

1. 1. Cut out your gene Cut out your gene of interest and put it in of interest and put it in a vectora vectoroDigest the plasmid and Digest the plasmid and DNA of interest with DNA of interest with samesame restriction enzyme (now restriction enzyme (now have compatible sticky have compatible sticky ends)ends)oSome plasmids will close Some plasmids will close up without geneup without geneoSome plasmids will get the Some plasmids will get the gene insertedgene inserted

How to Clone a Gene ContinuedHow to Clone a Gene Continued

2. Transforming Bacteria (putting the gene 2. Transforming Bacteria (putting the gene of interest with the plasmid into the cell) of interest with the plasmid into the cell)

Artificial TransformationArtificial Transformationo Need competent cells (in exponential growth)Need competent cells (in exponential growth)o Positive ions make cell membranes permeable Positive ions make cell membranes permeable

to DNAto DNAo Can also use electroporation, heat shockCan also use electroporation, heat shocko Can inject the DNACan inject the DNA

Gene Cloning ContinuedGene Cloning Continued

3. Selecting cells that have the gene of 3. Selecting cells that have the gene of interestinterest

Need to know if the gene is inserted into the Need to know if the gene is inserted into the plasmid and if the plasmid is inserted into the cellplasmid and if the plasmid is inserted into the cell

A. To see if colony has the plasmid - Use a plasmid that A. To see if colony has the plasmid - Use a plasmid that has antibiotic resistance gene – if plasmid is has antibiotic resistance gene – if plasmid is inserted, it will grow on antibiotic (like ampinserted, it will grow on antibiotic (like ampr r – and – and grow on amp)grow on amp)

B.B. To see if plasmid containing colonies have To see if plasmid containing colonies have plasmids with the gene of interest:plasmids with the gene of interest:

o Use restriction enzymes that cuts in the middle of a color Use restriction enzymes that cuts in the middle of a color gene like the gene like the ββ galactosidase gene – if inserted the galactosidase gene – if inserted the colonies will be whitecolonies will be white

Selection of cells with gene of Selection of cells with gene of interest continuedinterest continued

o Look for protein products by activity or Look for protein products by activity or antibodiesantibodies

o Make probes (short pieces of DNA or RNA Make probes (short pieces of DNA or RNA that will hybridize (base pair) with the gene that will hybridize (base pair) with the gene of interest – must be radioactive or of interest – must be radioactive or fluorescent.fluorescent.

o Transfer some cells from each colony to filter Transfer some cells from each colony to filter paper, probe, and then match up coloniespaper, probe, and then match up colonies

Technology #2 – Creating a Technology #2 – Creating a Genomic LibraryGenomic Library

1.1. Cut whole genome with restriction enzymes – makes Cut whole genome with restriction enzymes – makes sticky ends sticky ends

2.2. Cut plasmids with same restriction enzyme to make Cut plasmids with same restriction enzyme to make matching sticky matching sticky

3.3. Mix the 2 together to get a bunch of plasmids – each Mix the 2 together to get a bunch of plasmids – each with a piece of the genomewith a piece of the genome

Problems with Genomic LibrariesProblems with Genomic Libraries

1.1. There are many random fragments There are many random fragments

2.2. Must find the correct gene out of all of the plasmidsMust find the correct gene out of all of the plasmids

3.3. Contains introns that bacteria can’t transcribeContains introns that bacteria can’t transcribe

Use same restriction enzyme

to cut genomic DNA and plasmids to make matching

sticky ends

Making a Genomic Library

Technology #3: Creating a Technology #3: Creating a cDNA LibrarycDNA Library

1. Create the cDNA (complementary DNA)

Collect all of the mRNA from a cell

Use the enzyme reverse transcriptase (from retroviruses) to copy the mRNA into ds DNA

1. Cut cDNA with restriction enzymes, cut plasmids with same r.e. and mix together

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

due to primers (must know some sequences due to primers (must know some sequences flanking the gene)flanking the gene)

No need to isolate the gene firstNo need to isolate the gene first 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 Can use to amplify a gene of interest before Can use to amplify a gene of interest before

making a library so there is a higher making a library so there is a higher concentration of that gene in the libraryconcentration of that gene in the library

PCRPCR

1.1. Make primers complementary to the ends of Make primers complementary to the ends of the target sequencethe target sequence

2.2. Heat denature the DNA (96Heat denature the DNA (9600))

3.3. Cool DNA – primers stick (50Cool DNA – primers stick (5000))

4.4. Heat a little and let DNA polymerase copy the Heat a little and let DNA polymerase copy the ds DNA (72ds DNA (7200))

5.5. Heat denature againHeat denature again

6.6. CoolCool

7.7. Copy, repeat, repeat, repeatCopy, repeat, repeat, repeat

8.8. 30 cycle makes 200 million copies30 cycle makes 200 million copies

Double strand cDNA

AAAAA

TTTTTRT

AAAAA

TTTTTRT

RTAAAAA

TTTTT

Oligo dT primer is bound to mRNA

Reverse transcriptase

(RT) copies first cDNA strand

Reverse transcriptase digests and

displaces mRNA and copies

second strand of cDNA

Conversion of mRNA to cDNA by Reverse Transcription

RT PCRRT PCR

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

First round of cDNA

synthesis (4 strands)

Taq

Taq

Electrophoresis – Separation of Electrophoresis – Separation of molecules based on electrical molecules based on electrical

charge and sizecharge and sizeUses:Uses:

Determine the size of a fragmentDetermine the size of a fragment Purify plasmidsPurify plasmids Identify genes through hybridization/ Diagnosis Identify genes through hybridization/ Diagnosis

of genetic diseaseof genetic disease Sequencing of a geneSequencing of a gene RFLP or VNTR analysis RFLP or VNTR analysis

Paternity testingPaternity testing ForensicsForensics Chromosome mappingChromosome mapping

DNA ElectrophoresisDNA Electrophoresis Cut up DNA with restriction enzymesCut up DNA with restriction enzymes Load solution of cut up DNA into a well of an Load solution of cut up DNA into a well of an

agarose gel (porous gel that acts as a sieve)agarose gel (porous gel that acts as a sieve) Apply an electrical current to gelApply an electrical current to gel DNA is negatively charged so it moves to the DNA is negatively charged so it moves to the

positive pole.positive pole. Since the gel is Since the gel is

porous – the smallerporous – the smaller

the piece of DNA –the piece of DNA –

the faster it movesthe faster it moves

so it separates by sizeso it separates by sizeHow to pour, load, and run a gel

Electrophoresis after restriction Electrophoresis after restriction digestiondigestion

TATCTGGAAGTGGTACC GGAATCTACCGGTATCTGGAAGTGGTACC GGAATCTACCGG

TATCCGGAAGTGATACCGGAATCTACCGGTATCCGGAAGTGATACCGGAATCTACCGG

TATCCGGAAGTGGTATCGGAATCTACCGGTATCCGGAAGTGGTATCGGAATCTACCGG

Circular forms of DNA migrate in agarose distinctly differently from linear DNAs of the same mass. Typically uncut plasmids will appear to migrate more rapidly than the same plasmid when linearized. Additionally, most preparations of uncut plasmid contain at least two topologically-different forms of DNA, corresponding to supercoiled forms and nicked circles. The image to the right shows an ethidium-stained gel with uncut plasmid in the left lane and the same plasmid linearized at a single site in the right lane.

Plasmid PurificationPlasmid PurificationWhat a cut vs. uncut plasmid What a cut vs. uncut plasmid

looks likelooks like

Gene IdentificationGene IdentificationIf the DNA is purifiedIf the DNA is purified

Cut the DNACut the DNARun gel (DNA runs toward the + pole)Run gel (DNA runs toward the + pole)Stain if have purified geneStain if have purified gene

CCGCCG↓CGGTAGGAAC CCACGGTAGGAAC↓CGGTAGGAAC CCACGGTAGGAAC

________

________

________

Gene IdentificationGene IdentificationUsing Genomic DNAUsing Genomic DNA

Cut the DNACut the DNARun gel (DNA runs toward the + pole)Run gel (DNA runs toward the + pole) If stained the gel – big smear of DNA because If stained the gel – big smear of DNA because

so many bandsso many bandsSouthern Blot and ProbeSouthern Blot and ProbeProbe = ATCCTTProbe = ATCCTT

CCGCCG↓CGGTAGGAAC CCACGGTAGGAAC↓CGGTAGGAAC CCACGGTAGGAAC

________

________

Southern BlottingSouthern Blotting

Denature DNA in gelDenature DNA in gelTransfer to Nitrocellulose paper by Transfer to Nitrocellulose paper by

capillary actioncapillary actionProbe with labeled probesProbe with labeled probesWash non-specific probe off of paperWash non-specific probe off of paperExpose to filmExpose to filmCan see if a DNA sequence is there, how Can see if a DNA sequence is there, how

many fragments, size of fragmentsmany fragments, size of fragments

Stain vs. Southern Blot with Stain vs. Southern Blot with Genomic DNAGenomic DNA

Bacterial DNA Cut with a Bacterial DNA Cut with a Restriction EnzymeRestriction Enzyme

Left is stained/ Right is Blotted and ProbedLeft is stained/ Right is Blotted and Probed

Other Blotting TechniquesOther Blotting Techniques

Northern – same but using RNA instead of Northern – same but using RNA instead of DNADNA

Western Blotting – electrical transfer of Western Blotting – electrical transfer of proteins to paper and then using proteins to paper and then using fluorescently or radioactively tagged fluorescently or radioactively tagged antibodies to identify proteinantibodies to identify protein

Alternative for Genetic Disease Alternative for Genetic Disease DiagnosisDiagnosis

Can diagnose diseases without gels using Can diagnose diseases without gels using RT-PCRRT-PCRCollect cells – do PCR of a particular geneCollect cells – do PCR of a particular geneSequence the gene to see if it is normal or Sequence the gene to see if it is normal or

mutatedmutatedAside – can find presence of infectious Aside – can find presence of infectious

viruses this way – collect blood or cells viruses this way – collect blood or cells and PCR viral genes – run on gel or and PCR viral genes – run on gel or sequence to see if presentsequence to see if present

RFLP or STR – Paternity Testing RFLP or STR – Paternity Testing and DNA Fingerprintingand DNA Fingerprinting

RFLP – RFLP – restriction fragment length polymorphismsrestriction fragment length polymorphisms

Same as a Southern Blot but usually Same as a Southern Blot but usually probe for non-coding regions of DNA that probe for non-coding regions of DNA that are highly variable (usually these regions are highly variable (usually these regions are more variable than actual genes)are more variable than actual genes)

Usually use multiple probesUsually use multiple probesSTR (short tandem repeats)STR (short tandem repeats)Found to be the most variable among Found to be the most variable among

humans – makes fragments of different humans – makes fragments of different sizes if different number of repeats in sizes if different number of repeats in satellite DNAsatellite DNA

DNA Fingerprinting

Must do stats for each probe – what is the % of people that carry each restriction pattern for that

probe

The more probes, the more sure you can be that the pattern fits only one person

6 probes is very good

Probe 1 – 1/10 exhibit this pattern

Probe 2 – 1/20

Probe 3 – 1/100

Probe 4 – 1/10

Probe 5 – 1/50

Probe 6 – 1/5

What is the chance that this profile can belong to another person?

1/50,000,000

Combined DNA Index System

Run by the FBI

Has over 5 million convicted offender DNA profiles

Mandatory to have DNA profile in CODIS if involved in a homicide or

sex crime

Uses 13 different loci to look at 13 different areas for differences in

their STR

Creates a unique pattern – 1 in 10 billion have matching pattern

Paternity TestingPaternity Testing

Run standard RFLP analysis Run standard RFLP analysis using 3-4 probesusing 3-4 probes

The child gets 2 copies of every The child gets 2 copies of every gene – 1 from each parentgene – 1 from each parent

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

Alleged fathers can be excluded Alleged fathers can be excluded but many times if the pattern but many times if the pattern matches – there is only a 99% matches – there is only a 99% chance that he is the fatherchance that he is the father

Usually about 1/100 people Usually about 1/100 people have that same patternhave that same pattern

Sequencing – Sanger MethodSequencing – Sanger Method

Cut DNA with restriction enzymesCut DNA with restriction enzymes Divide into 4 reaction tubes with all the stuff for Divide into 4 reaction tubes with all the stuff for

replication – (DNA polymerase, ligase, replication – (DNA polymerase, ligase, triphosphate nucleotides)triphosphate nucleotides)

Add ddATP to 1 tube, ddTTP to 1 tube, ddGTP Add ddATP to 1 tube, ddTTP to 1 tube, ddGTP to 1 tube, ddCTP to another tubeto 1 tube, ddCTP to another tube

As DNA copies, eventually each nucleotide will As DNA copies, eventually each nucleotide will be “labeled”be “labeled”

Longest fragments at top, smallest at bottom, Longest fragments at top, smallest at bottom, read from the bottom upread from the bottom up

Can do in one tube with fluorescently labeled Can do in one tube with fluorescently labeled ddnucleotides – A,T,G,C a different colorddnucleotides – A,T,G,C a different color

Instruments for SequencingInstruments for Sequencing

New InstrumentsNew Instruments

Microarray Analysis Microarray Analysis Gives the ability to compare gene expression between:Gives the ability to compare gene expression between: Different tissuesDifferent tissues Different speciesDifferent species Different stages of developmentDifferent stages of development Cancer vs. non-cancerCancer vs. non-cancer Healthy vs. diseased tissueHealthy vs. diseased tissue

Procedure:Procedure: Make the microarray plate (a robot attaches single stranded Make the microarray plate (a robot attaches single stranded

pieces of DNA to a glass plate including every gene in the pieces of DNA to a glass plate including every gene in the genome)genome)

Make cDNA from all mRNA in each of the two cell types you Make cDNA from all mRNA in each of the two cell types you want to comparewant to compare

Make the cDNA ss and attach a red tag to the cDNA from one Make the cDNA ss and attach a red tag to the cDNA from one cell type and a green tag to the cDNA from the other cell typecell type and a green tag to the cDNA from the other cell type

Add the tagged cDNA’s to the plate and let them hybridizeAdd the tagged cDNA’s to the plate and let them hybridize If the spot on the plate is red – it is only expressed in that cell If the spot on the plate is red – it is only expressed in that cell

type, if it is green – only expressed in the other cell type, yellow type, if it is green – only expressed in the other cell type, yellow – expressed in both (can even tell concentration differences)– expressed in both (can even tell concentration differences)

Affix ss genes to Affix ss genes to glass plateglass plate

Represents all Represents all genes of genomegenes of genome

A scanner reads the amount of red

fluorescence and green fluorescence

separately so you can even tell slight

differences in gene expression between

the two samples

In-Vitro Mutagenesis and In-Vitro Mutagenesis and TransgenicsTransgenics

Try to get a handle on the function of the Try to get a handle on the function of the genegene

Can change a gene and put it in a cell and Can change a gene and put it in a cell and see the effect on the cellsee the effect on the cell

Can mutate a gene in an embryonic cell Can mutate a gene in an embryonic cell and have it try to develop and see what and have it try to develop and see what happens in development or in adulthoodhappens in development or in adulthood

Cloning of OrganismsCloning of Organisms Destroy DNA in eggDestroy DNA in egg Put DNA from an adult somatic cell into the eggPut DNA from an adult somatic cell into the egg Put the egg into a female host uterus and Put the egg into a female host uterus and

develop into a new organismdevelop into a new organism Most are messed up, develop diseases, or die Most are messed up, develop diseases, or die

prematurelyprematurely Using DNA with epigenetics (methylation, Using DNA with epigenetics (methylation,

acetylation patterns) of an adult cell – not of an acetylation patterns) of an adult cell – not of an embryo – affects gene expression!embryo – affects gene expression!

Embryo farms – grow balls of cells that are Embryo farms – grow balls of cells that are clones to use for stem cells to grow new parts – clones to use for stem cells to grow new parts – ethical?????ethical?????

Stem Cell ResearchStem Cell Research Pluripotent embryonic stem cells can become Pluripotent embryonic stem cells can become

any kind of cellany kind of cell Adult stem cells become a particular kind of cell Adult stem cells become a particular kind of cell

or one of a few kinds of cellsor one of a few kinds of cells Working on taking normal differentiated cells Working on taking normal differentiated cells

and turning them back into stem cells so can and turning them back into stem cells so can use them to make different kinds of cells (ex. use them to make different kinds of cells (ex. Skin becomes stem cell becomes a neuron)Skin becomes stem cell becomes a neuron)

Ethical issues? – where are we getting them Ethical issues? – where are we getting them from?from?

Found some stem cells in brain – can make Found some stem cells in brain – can make some new neurons!some new neurons!