How to characterize a single piece of DNA

- Isolate a small fragment of DNA

- Insert DNA into plasmid (or phage vector)

-Transform recombinant DNA molecule into bacteria

-Amplify DNA by culturing transformed bacteria

-Use transformants for variety of purposes (e.g.expression studies,sequencing, mutational analysis, etc.)

-Select for transformants

LE 20-2Bacterium

Bacterialchromosome

Plasmid

Gene inserted intoplasmid

Cell containing geneof interest

Gene ofinterest DNA of

chromosome

RecombinantDNA (plasmid)

Plasmid put intobacterial cell

Recombinantbacterium

Host cell grown in cultureto form a clone of cellscontaining the “cloned”gene of interest

Protein expressedby gene of interest

Protein harvested

Gene ofinterest

Copies of gene

Basicresearchon gene

Basicresearchon protein

Basic research andvarious applications

Gene for pestresistance insertedinto plants

Gene used to alterbacteria for cleaningup toxic waste

Protein dissolvesblood clots in heartattack therapy

Human growth hor-mone treats stuntedgrowth

Restriction Enzymes Used to Make Recombinant DNA

• Bacterial restriction enzymes

– cut DNA molecules at specific DNA sequences called restriction sites

LE 20-3Restriction site

DNA 53

35

Restriction enzyme cutsthe sugar-phosphatebackbones at each arrow.

One possible combination

DNA fragment from anothersource is added. Base pairingof sticky ends producesvarious combinations.

Fragment from differentDNA molecule cut by thesame restriction enzyme

DNA ligaseseals the strands.

Recombinant DNA molecule

Sticky end

EcoRI

MemorizeEcoRI restriction site

palindrome

Catalyzes phosphodiesterbond between5’ phosphate &3’ hydroxyl group of sugar

Ligation

Do restriction digests and ligations always work?

What are the other possible undesirable outcomes?

Clever way to select for recombinant clones

Plasmid

contains LacZ gene-->-galactosidase

X-gal (substrate:one product is blue)

Blue colonies

Restriction site in LacZ gene

if insert DNA fragment -galactosidase

X-gal

White colonies

What is a common strategy to select for transformed bacteria?

Grow bacteria on antibiotic: only plasmid carriers will survive

LE 20-4_3

Isolate plasmid DNAand human DNA.

Cut both DNA samples withthe same restriction enzyme.

Mix the DNAs; they join by base pairing.The products are recombinant plasmidsand many nonrecombinant plasmids.

Bacterial cell lacZ gene(lactosebreakdown)

Humancell

Restrictionsite

ampR gene(ampicillinresistance)

Bacterialplasmid Gene of

interest

Stickyends

Human DNAfragments

Recombinant DNA plasmids

Introduce the DNA into bacterial cellsthat have a mutation in their own lacZgene.

Recombinantbacteria

Plate the bacteria on agarcontaining ampicillin and X-gal.Incubate until colonies grow.

Colony carrying non-recombinant plasmidwith intact lacZ gene

Colony carryingrecombinantplasmid withdisrupted lacZ gene

Bacterialclone

Different goals in creating recombinant clones

1. To examine/utilize the structure and function of a single piece of DNA.

2. To package small pieces of an entire genome: genomic DNA library

To have available all the sequences in the genome for examination and use.

LE 20-6

Bacterialclones

Recombinantplasmids

Recombinantphage DNA

or

Foreign genomecut up withrestrictionenzyme

Phageclones

Plasmid library Phage library

DNA libraries created using plasmids and phage and bacterial hosts

Note: practical limit on the size of DNA cloned into a vectors (plasmid: 5-10 kbp, phage: 45 kbp)

Characterization of DNA by Size

Agarose Gel Electrophoresis

Digest DNA with restriction enzymes

Load DNA into wells of agarose gel

Apply electric current to fractionate DNA fragments by size

In electric field with positive and negative poles, which polewill DNA be attracted to? Why?

How to distinguish one DNA molecule from another?

LE 20-8

Cathode

Powersource

Anode

Mixtureof DNAmoleculesof differ-ent sizes

Gel

Glassplates

Longermolecules

Shortermolecules

-DNA stainedwith fluorescentdye (ethidium bromide)

-DNA fluoresces upon exposure toultraviolet (UV) light

How would you determine whether a particular gene or DNA sequence is present in your cloned DNA?

Southern Blot

LE 20-10

DNA + restriction enzyme Restrictionfragments

Normal-globinallele

Sickle-cellallele

Heterozygote

Preparation of restriction fragments. Gel electrophoresis. Blotting.

Nitrocellulosepaper (blot)

Gel

Sponge

Alkalinesolution

Papertowels

Heavyweight

Hybridization with radioactive probe.

Radioactivelylabeled probefor -globingene is addedto solution ina plastic bag

Paper blot

Probe hydrogen-bonds to fragmentscontaining normalor mutant -globin

Fragment fromsickle-cell-globin allele

Fragment fromnormal -globinallele

Autoradiography.

Film overpaper blot

Southern Blot AnalysisLabeled nucleic acid probe:RNA or DNA

Why are globin DNA fragments different in size?

LE 20-9Normal -globin allele

175 bp 201 bp Large fragment

Sickle-cell mutant -globin allele

376 bp Large fragment

Ddel Ddel Ddel Ddel

Ddel Ddel Ddel

Ddel restriction sites in normal and sickle-cell alleles of-globin gene

Normalallele

Sickle-cellallele

Largefragment

376 bp201 bp175 bp

Electrophoresis of restriction fragments from normaland sickle-cell alleles

Restriction enzyme

Restriction Fragments Length Polymorphisms (RFLP)

- useful in detecting disease alleles

-forensicsto identify individualsno two individuals are alike

(exception?)

LE 20-17Defendant’sblood (D)

Blood from defendant’sclothes

Victim’sblood (V)

Do the RFLPs suggest the defendant was in contact withthe victim?

By themselves,do RFLPSprove she’s guiltyof assault?

Genomics and Molecular Techniques

• Characterization of entire genomes

• Human Genome Project (HPG):• ambitious goal to sequence the entire human

genome (initiated 1990; mostly complete 2003)

• Other genomes also sequenced

• Evolutionary relatedness of key interest->sequence comparison

Ch 20

LE 20-11

Cytogenetic map

Genes locatedby FISH

Chromosomebands

Geneticmarkers

Genetic (linkage)mapping

Physical mapping

Overlappingfragments

DNA sequencing

Stepsin genomemapping

(chromosome map)

DNA Sequencing

• Short DNA fragments sequenced by dideoxy chain-termination method

LE 20-12

DNA(template strand)

5

3

Primer3

5

DNApolymerase

Deoxyribonucleotides Dideoxyribonucleotides(fluorescently tagged)

3

5DNA (templatestrand)

Labeled strands3

Directionof movementof strands

Laser Detector

DNA chainterminators

• Other approach to genome sequencing:

– Shotgun method• Sequence random fragments of DNA• Computer program orders overlapping fragments

into single continuous sequence

LE 20-13

Cut the DNA from many copies of an entire chromosome into overlapping frag-ments short enough for sequencing

Clone the fragments in plasmid or phagevectors

Sequence each fragment

Order the sequences into one overall sequence with computer software

• Genome organization• Gene expression patterns in response to

- environmental change e.g.pollution, global

warming-Development

embryogenesis->senescence

-Disease/Health

Can we learn important information from the genome sequence?

Computer Analysis: Key Tool

Bioinformatics

-analysis and storage of biological data by computing techniques

-key to management & analysis of huge data sets

Example: Identification of proteins coding sequences (ORF)in genomes

agatactagcagctctttcgagcatcagcatcaccgatgcatcgatcacgcgctgtttg…

Think of a sequence feature that a program could search for to identify ORFs.

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