announcements lab notebooks due monday by 5 no ch. 9 part 2 homework we will have lab next week....

Announcements

Lab notebooks due Monday by 5 No Ch. 9 Part 2 homework We will have lab next week. It just isn’t a typical

lab-discussing individual projects

Ch. 9 Part 2: Biotechnology and Recombinant DNA

Human Genome Project produced great advances• Rapid growth in field of genomics• Sequencing of numerous organisms• Spawned new field of bioinformatics to analyze data• Allows determination of amino acid sequences

• Comparisons of different proteins, various organisms• Evolutionary relatedness of organisms• Technology so efficient that new Human Microbiome

Project aims to determine biological diversity of normal microbiota

Toolkit: DNA Sequencing-Why It Is Important

Dideoxy chain termination most common method• Automated and fast• In vitro DNA synthesis• Template DNA• DNA polymerase• Primer• Deoxynucleotides

• dNTPs• Dideoxynucleotides

• ddNTPs

Toolkit: DNA Sequencing How It Works

Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

Final product

Primer anneals to complementary sequence, serving as the nucleic acidfragment to which DNA polymerase can add nucleotides.

DNA polymerase Nucleotide

Primer is added to single-stranded DNA.

Primer

5′

T T G C C GT A

T A C C G CG C C TT A A A T T G A A TT G G C C G G G A A T T T A A C T

T T G C C G T A C C G G C

T G G C C G

T G G C C GT T G C C G T A C C G G C C C T T A A A T T G A A T

5′

5′3′

3′

3′

3′ G CAT

C C T T A A A T T G A A T

5′ 3′

3′5′

5′

Dideoxy chain termination most common method (continued…)• Dideoxynucleotides (ddNTPs)

• Serve as chain terminators– Lack 3′OH

– Synthesis stops

– Yields mixture of DNA of different lengths

– Denature DNA

– Separate ssDNA viaelectrophoresis

– Read fluorescent labelon ddNTPs to obtainsequence


Incorporation of adeoxynucleotide (dNTP)elongates the chain. Asubsequent nucleotide canbe added to the 3′OH.

Incorporation of adideoxynucleotide (ddNTP);the ddNTP lacks a 3′OH.

Chain elongation isterminated. No additionalnucleotides can be addeddue to the lack of a 3′OH.

PO4 OH

5′

PO4

5′

OHH PO4

X5′3′

5′

3′

5′

5′

3′

3′

H

OH

3′OH

Toolkit: DNA Sequencing How It Works

Gel electrophoresis• Laser reads color• Determines sequence


1

2 3

Decreasin

g frag

men

t size

Key ingredients in the reaction:• Primer and template (shown annealed)

A T A G C T A C C T A G

• DNA polymerase• Deoxynucleotides (dATP, dTTP, dGTP, dCTP)• Fluorescently labeled dideoxynucleotides (ddATP, ddTTP, ddGTP, ddCTP; a very small amount of each)

The fragments are denatured, and the singlestrands then separated by gel electrophoresis.The color of the fluorescent marker indicatesthe terminating dideoxynucleotide.Results:

T A T C G A T G G A T C*A T A G C T A C C T A G

T A T C G A T G G A T*A T A G C T A C C T A G

T A T C G A T G G A*A T A G C T A C C T A G

T A T C G A T G G*A T A G C T A C C T A G

T A T C G A T G*A T A G C T A C C T A G

T A T C G A T*A T A G C T A C C T A G

T A T C G A*A T A G C T A C C T A G

T A T C G*A T A G C T A C C T A G

T A T C*A T A G C T A C C T A G

T A T*A T A G C T A C C T A G

T A*A T A G C T A C C T A G

T*A T A G C T A C C T A G

Chain elongation is terminated randomlywhen DNA polymerase incorporates adideoxynucleotide (indicated by a coloredbox and an asterisk).Products of the reaction:

Toolkit: DNA Sequencing

Allows amplification to millions of copies of DNA• Matter of hours• Products can be visualized

via gel electrophoresis• Allows detection of

specific sequences

• Can detect organismswithout culturing

– E.g., pathogens

• Requires template DNA, polymerase, primers, deoxynucleotides(dATP, dGTP, dCTP, dTTP)

Toolkit: Polymerase Chain Reaction (PCR)

Conclusion:Patient A is positive (infected);Patient B is negative.

DNA from Patient A DNA from Patient B

PCR amplifiesspecific sequence

of interest.

No DNA amplified

Patient A Patient B

Gel electrophoresis ofPCR amplified samples


Three-step amplification cycle• DNA denatured by heating (~95°C)• Temperature lowered (~50°C) to

allow primers to anneal• Temperature raised (~70°C) to

allow DNA synthesis• DNA doubled in each cycle, so

exponential increase• Heat-stable Taq polymerase from

Thermus aquaticus critical


1

3

2

Heating to 95°C denatures DNA.

Cooling to 50°C allows the addedprimers to anneal to the single-stranded templates.

DNA synthesis occurs when thetemperature is raised to 72°C.

The products of one 3-step cycle of PCR

Region of DNA to be amplified

5′3′

3′

5′

3′

3′

5′

3′

5′5′

3′

5′5′

5′

3′

5′

3′

5′3′

5′

3′

Primer

5′

3′

3′5′

5′

3′

5′

Primer

3′5′3′

3′


Primers define length of PCR product



1

2

3

Mid-length strands are synthesizedfrom the full-length templates. Thesites to which the primers annealeddetermine the 5′ ends of these strands.

When the mid-length strands madein the reaction above are used astemplates, short strands aregenerated. The 3′ ends of thefragments are complementary to the5′ ends of the primers.

Outcome of cycle 1(as well as subsequent cycles)



Target DNA

Template

PrimerMid-lengthfragment

Mid-lengthfragment

Template

Short fragment

Cycle 1 mid-lengthfragment (template)

Cycle 1 mid-lengthfragment (template)

Short fragment

Short fragment

Cycle 2 shortfragment (template)

Cycle 2 shortfragment (template)

Short fragment

When short strands made in thereaction above are used astemplates, like-sized strands aregenerated. This is the double-stranded target molecule that will beamplified exponentially.

5′

5′

3′

3′

5′

3′

3′

3′

3′

5′

3′

Produced when full-length molecule is used as a template

Produced when mid-length molecule is used as a template

Produced when target fragment is used as a template

5′

3′

5′

3′

3′

5′

3′

5′

5′

Primer

5′

5′

3′

5′

Exponential amplification of DNA• Increasing numbers

of product of correctly defined length

• ~30 cycles typical


End of cycle 5End of cycle 4End of cycle 3End of cycle 2End of cycle 1 Time 0

(2)

(8)

(22)


Gel Electrophoresis of PCR Product

DNA probes locate specific nucleotide sequence• Probe is single-stranded piece of DNA

• Will hybridize to complementary sequence• Labeled with marker• Numerous different

probe technologies

Toolkit: Probe Technologies-Why Important


LabelProbe

Probe is added to single-stranded DNA that has been attached to asolid surface.

Probe anneals to complementary sequence. Because of the label itcarries, its location can easily be determined.

T

G

G G C C G

T T C C G T A C C G G C C C T T A A A T T G A A T

T G G C C GT T G C C G T A C C G G C C T T A A AT TG ATAC

3′

5′ 3′

5′

5′3′

3′

Colony Blotting• Allows detection of colonies that have specific

sequence of DNA• Commonly used to identify which clones in a

collection contain sequence of interest

Toolkit: Probe Technologies

Nylon membrane

Probe is added thatbinds to DNA ofinterest.

The membrane issoaked in an alkalinesolution to lyse the cellsand denature theirDNA.

Probe bound to DNA

Colonies on an agarplate.

Colonies are transferredin place (“blotted”) to anylon membrane.

By locating thepositions to whichprobe has bound,colonies that have theDNA of interest can belocated.

21 3 4 5


Fluorescence in situ Hybridization (FISH)• Probe hybridizes to nucleotide sequences in intact cells

fixed to microscope slide• Cells visualized via fluorescence microscope• Probe often binds to rRNA

sequences since numerous• Rapid identification in

specimen without culturing• Related organisms or

specific species


DNA Microarrays• Studies of gene expression• Also used to detect DNA

sequences• mRNA is isolated, converted

to labeled cDNA, hybridized to array

• Array has numerous short probes specific to each gene of interest

• Allows comparisons of conditions


announcements lab notebooks due monday by 5 no ch. 9 part 2 homework we will have lab next week....

Documents

annealeda t

randomlywhen dna polymerase

singlestranded dna

chain terminatorslack

mcgrawhill companies

common methodautomated

field of genomicssequencing

new human microbiome