Chapter 12DNA Technology and the Human Genome

Do Now: 10 minutesStudy Technique

• Skim chapter opening pg 230-231• 1) Define bold words and look at pictures• 2) Make a quick outline of main points with at

least 3 spaces in between in one color• 3) Use a different color to fill in under each

section with details from the book• 4) In a different color come up with two

questions, one easy and one difficult

Should look like this….

• Recombinant DNA technology: combining genes from different sources• DNA technology: methods for studying and manipulating DNA• Genomics: Study of genomes based on sequence

– Bioinformatics: using math to analyze bio data– Proteomics: study of proteins

• Bacteria studies– Joined by pilli (male) passing DNA to “female”– Respond to environment– Lederburg and Tatum: experiments that mixed genes in E. coli (Recombinant

DNA technology– DNA technology

• Introns discovered by this• Lead in to HGP

• Human Genome project (more than just humans)– Mapping genomes lead to genomics, bioinformatics, and proteomics– Scientists engineer bacteria for practical purposes (make pesticides, cancer

drugs, AIDS vaccine)– Ethical issues on health

What might Ms. Bell Ask…

• Easy: How do bacteria have sex?• Hard: Why is this advantageous for bacteria?

• Easy: Describe the difference between genomics, proteomics, and bioinformatics

• Hard: Why are people concerned with recombinant DNA technology? Explain a hypothetical situation.

12.1 In nature, bacteria can transfer DNA in three ways

• Gene transfer– DNA from one to another bacteria– Non-reproductive – new combination– DNA= closed loop w/protein

• Three mechanisms– 1) Transformation: uptake from surrounding

• Griffith experiment: harmless bacteria take up piece from deadly and became deadly….

– 2) Transduction: phage– 3) Conjugation: Male donor, pili

• Female recipient• Cytoplasmic bridge

• New DNA may integrate into recipient

• Question… Which type of gene transfer allows one copy of DNA to peel off and transfer to recipient?

12.2 Bacteria plasmids can serve as carriers for gene transfer

• F factor– Genes for conjugation– Also contains origin of replication– Acts as the leading end of transferred chromosome

• Plasmid– Small circular DNA that contains origin of replication– Some contain F factor– Vector s: Can carry genes other than F factor

• R plasmid– Contain enzymes that destroy antibiotics like penicillin or

tetracycline– Act as useful vectors in genetic engineering

LE 12-02Restriction enzyme

recognition sequence

Restriction enzymecuts the DNA intofragments

Addition of a DNAfragment fromanother source

DNA G CA AT T

T

T

CG

GC

C

CGG

AA

AA

AAT TT TAA

T

T

Sticky end

Two (or more)fragments sticktogether bybase-pairing

DNA ligasepastes the strand

Recombinant DNA molecule

GG

GG C

CC

CAAAA

AATT

TTTT

T T AA

C T T A A GHow restriction enzymes work…

LE 12-01-3

Bacterialchromosome

Plasmid

BacteriumPlasmidisolated

Recombinant DNA(plasmid)

Recombinantbacterium

Copies of gene

Clone of cellsGene for pestresistanceinserted intoplants

Gene used to alter bacteriafor cleaning up toxic waste

Protein used to dissolve bloodclots in heart attack therapy

Copies of protein

Cell multiplies withgene of interest

Protein used tomake snowform at highertemperature

Plasmid put intobacterial cell

DNAGene ofinterest

Gene insertedinto plasmid

DNAisolated

Cell containing geneof interest

12.3 Plasmids are used to customize bacteria: An overview

• 1) plasmid isolated• 2) DNA of interest is obtained• 3) DNA inserted • 4) Bacteria uptakes plasmid• 5) genetically engineered, recombinant bacteria

cloned

• Challenge: How can R plasmids be helpful in terms of recombinant DNA technology?

LE 12-03Isolate DNAfrom two sources

Cut both DNAswith the samerestriction enzyme

Mix the DNAs;they join bybase-pairing

Add DNA ligaseto bond the DNA covalently

Recombinant DNAplasmid

E. coli

Plasmid

Human cell

Sticky endsGene V

DNA

Gene V

Put plasmid into bacteriumby transformation

Recombinantbacterium

Clone the bacterium

Bacterial clone carrying manycopies of the human gene

LE 12-04

Recombinantplasmid

Bacterialclone

Plasmid library

Phageclone

Phage library

Recombinantphage DNA

Genome cut up withrestriction enzyme

or

12.7 Reverse Transcriptase helps make genes for cloning

• Do Now: Turn to page 236 section 12.7• 1) Read the section• 2) Redraw the picture• 3) Describe cDNA and the process of making a

cDNA• 4) Why might cDNA of insulin might work

better in E.coli than trying to use a normal gene?

LE 12-05

Cell nucleus

mRNA

Test tube

DNA ofeukaryoticgene

Reverse transcriptase

cDNA strand

cDNA of gene(no introns)

RNAtranscript

Exon Exon ExonIntron Intron

Transcription

RNA splicing(removes introns)

Isolation of mRNAfrom cell and additionof reverse transcriptase;synthesis of DNA strand

Breakdown of RNA

Synthesis of secondDNA strand

12.8 Nucleic acid probes identify clones carrying specific genes

• It is difficult to tell if clone contains ________________________– Methods depends on base pairing

• Part of the DNA is known• Gene _________• Probe (radioactive) _________

– Steps: • 1) _________________________________• 2) Treat (heat or alkali) to ___________________• 3) Add radioactive probe (reality ___________________)• 4)Autoradiography (develop film to determine radioactivity)• 5) Compare to master plate (____________________)

LE 12-08

Radioactiveprobe (DNA)

Single-strandedDNA

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Base pairingindicates the gene of interest

A

A

T A

T

C

C C G

G

A

AT

CC

G

AA

A

AG

G

GG

CT

T

G

G

G

GC

C

C

A

A

A

T

T

T

T

C

C

TT

A T

12.9 DNA microarrays test for expression of many genes

• DNA microarrays (chips)– Used for______________________________– Measures gene expression at _______________– Steps• ______________________• mRNA used to make radioactive ________• Exposed to a chip

– Benefits• Thousands of genes tested at once• Each radioactive spot represents gene expression

LE 12-09

mRNAisolated

cDNA madefrom mRNA

cDNA appliedto wells

UnboundcDNA rinsedaway

Each well contains DNAfrom a particular gene

Fluorescentspot

DNA microarray

Nonfluorescentspot

cDNA

DNA of anexpressed gene

DNA of anunexpressed gene

Reverse transcriptaseand fluorescent DNAnucelotides

Actual size(6,400 genes)

12.10- 12.11 Gel electrophoresis:

Mixture of DNAmolecules of different sizes

Powersource

Gel

Completed gel

Longermolecules

Shortermolecules

12.12 The PCR method is used to amplify DNA sequences

• Polymerase chain reaction• Steps:

– _____________________– DNA is incubated

with____________________________________________

• Benefits:– Each cycle doubles DNA

amount– Takes hours – Can copy specific segment– Starting material does not

need to be purified

PCR: The good and the bad

• Even though it is faster, it cannot replace gene cloning in cells due to occasional errors that impose limits

• Uses:– ________________________– ________________________

12.13 Most of the human genome does not consist of genes

• Most of our DNA does not code for protein– 35,000 genes that code for protein, tRNA and

rRNA– 97% is non-coding

• Types of junk– __________and DNA between genes– Repetitive DNA

• Short repeats– Associated with ________________and ends of chromosomes

(chromosome structure)– Telomeres: at the end of chromosomes and related to cell death and

cancer– Cells that can regenerate telomeres can “_______________”

• Might deal with gene ___________________• Some nervous diseases caused by abnormal stretches of triplet

– Huntington's: _____ repeat in coding region

• Long Repeats: function unknown– Example: Transposons: genes that can jump from one location to the

next (researcher Barbara McClintock)» Cut and paste or ______, cut and paste (leaves sequence behind)» Natural mutagen to generate ________________» Implicated in some cancer

LE 12-11b

Longerfragments

1

x

Shorterfragments

w

y y

z

2

LE 12-12a

Defendant’sblood

Blood fromdefendant’s clothes

Victim’sblood

LE 12-13Cloned gene(normal allele) Insert normal gene

into virus

Viral nucleicacid

Retrovirus

Infect bone marrowcell with virus

Viral DNA insertsinto chromosome

Bone marrowcell from patient

Bonemarrow

Inject cellsinto patient

LE 12-14

InitialDNAsegment

Number of DNA molecules

1 2 4 8

12.4-12.6 Recombinant Lab

• Add picture from each….