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Genetics in the News

Genetics in the News Recombinant DNA Technology

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Page 1: Genetics in the News Recombinant DNA Technology

Genetics in the News

Page 2: Genetics in the News Recombinant DNA Technology

Recombinant DNA Technology

…artificial manipulation of DNA.

Page 3: Genetics in the News Recombinant DNA Technology

– isolate DNA,

– cut DNA into workable sized fragments,

– amplify the fragments for storage and subsequent

analysis,

– identify and isolate specific sequences,

– characterize by size genomic location and sequence.

Recombinant DNA Technology

Page 4: Genetics in the News Recombinant DNA Technology

Cutting the DNA

• ~3,100,000,000 base pairs per human haploid genome,

– average chromosome length is ~13 million base pairs,

• DNA is cut with bacteria-derived enzymes,

• The act of cutting is termed “digestion”.

Page 5: Genetics in the News Recombinant DNA Technology

Restriction Enzymes

…proteins that recognize specific, short nucleotide sequences and cut DNA at those sites,

…bacteria contain over 400 such enzymes and recognize and cut over 100 DNA sequences.

Page 6: Genetics in the News Recombinant DNA Technology

Bacteria Defense Mechanism

• restriction enzymes are expressed in bacteria in order to protect against invasion by viruses,

• bacterial protect their own DNA by chemically modifying recognition sites.

Page 7: Genetics in the News Recombinant DNA Technology

Palindromes

stir grits

no lemon, no melon

5’-------G-A-A-T-T-C--------3’

3’-------C-T-T-A-A-G--------5’

Page 8: Genetics in the News Recombinant DNA Technology

EcoRI

5’-------G A-A-T-T-C--------3’

3’-------C-T-T-A-A G--------5’

Page 9: Genetics in the News Recombinant DNA Technology

Sticky Ends

...single-stranded DNA overhangs resulting from restriction digestion.

Page 10: Genetics in the News Recombinant DNA Technology

EcoRI

5’-------G A-A-T-T-C--------3’

3’-------C-T-T-A-A G--------5’

Page 11: Genetics in the News Recombinant DNA Technology

RsaIRhodopseuomonas sphaeroides

5’-------G-T A-C--------3’

3’-------C-A T-G--------5’

Page 12: Genetics in the News Recombinant DNA Technology
Page 13: Genetics in the News Recombinant DNA Technology

Restriction Fragment Length Sizes(predicted)

- IF -

25% A 25% T 25% G 25% C

and

Random Distribution of Nucleotides

- THEN -

Distance between cut sites is equal to 4n bases,

(n = number of base pairs in the recognition site)

i.e. p(specific base) = .25

Page 14: Genetics in the News Recombinant DNA Technology

Average Restriction Fragment Length

n = 4, 256 base pairs

n = 6, 4096 base pairs

n = 8, 65.5 kb base pairs

Page 15: Genetics in the News Recombinant DNA Technology
Page 16: Genetics in the News Recombinant DNA Technology

Still Lots of Fragments

4 cutter: 3,000,000,000 bp ~256 bp = 12,000,000 fragments

6 cutter: 3,000,000,000 bp ~4096 bp = 700,000 fragments

8 cutter: 3,000,000,000 bp ~65.5 kb = 46,000 fragments

Page 17: Genetics in the News Recombinant DNA Technology

Ligation

…sticky ends with complementary base pairs can form hydrogen bonds,

…DNA ligase: an enzyme that catalyzes the reformation of the phosphodiester bonds.

Page 18: Genetics in the News Recombinant DNA Technology

Ligation

5’-------G A-A-T-T-C--------3’

3’-------C-T-T-A-A G--------5’

hydrogen bonds align,DNA ligase covalently links

Page 19: Genetics in the News Recombinant DNA Technology

DNA is Profligate

…sticky ends with complementary base pairs can form hydrogen bonds,

…with DNA from any species.

Page 20: Genetics in the News Recombinant DNA Technology

Cloning

…specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.

Page 21: Genetics in the News Recombinant DNA Technology

Requires a Vector…

...a specialized DNA sequence that can enter a living cell,

...signal its presence to an investigator,

... and provide a means of replication for itself and the foreign DNA it carries.

Cloning

…specialized DNA technology to produce multiple, exact copies of a single gene or other segment of DNA to obtain enough material for further study.

Page 22: Genetics in the News Recombinant DNA Technology

And, Vectors…

…contain unique restriction sites to facilitate the creation of recombinant DNA molecules,

... must also possess a distinguishing physical characteristic such as size or shape by which it can be purified away from the host cells genome.

Mike

Page 23: Genetics in the News Recombinant DNA Technology

Vectors

Plasmid E. coli up to 15 kb,

Phage E. coli up to 25 kb,

Cosmid E. coli up to 45 kb,

BAC E. coli 100-500 kb,

YAC Yeast 250-1000 kb.

Page 24: Genetics in the News Recombinant DNA Technology

Step 1…restriction digests and ligation of fragments into cloning vectors,

Page 25: Genetics in the News Recombinant DNA Technology

Cloning Step 2

…vector-insert recombinants are inserted in host cells,

– Transformation (via bacterial mechanisms);• plasmids• BACS• YACS

– Transduction (via virus mechanisms); • phage• cosmids

Page 26: Genetics in the News Recombinant DNA Technology

Cloning Step 3

...vectors contain selectable markers,

– only cells that contain vector DNA will survive selection,

– recombinant vectors can be discerned from empty vectors by additional markers.

Blue/White Cloning

Page 27: Genetics in the News Recombinant DNA Technology

Amplification

…if you were to grind me up with a giant mortar and pestle, and extract all of my DNA coding for a single gene, you’d get about 1 g of DNA,

…two liters of bacterial culture carrying my hemoglobin in a recombinant DNA vector, would yield an equivalent mass of DNA.

Page 28: Genetics in the News Recombinant DNA Technology

Assignment

• Read 10.1 and be prepared to clone a fragment of DNA using a plasmid as a vector.

Page 29: Genetics in the News Recombinant DNA Technology

B-PCR vs A-PCR

PCR, Polymeras Chain Reaction

Page 30: Genetics in the News Recombinant DNA Technology

TodayMore DNA Science

• DNA Amplification II,

– Polymerase Chain Reaction,– 6.7, pp. 237-239,

• Gel Electrophoresis,

– Southern Blots, Northern Blots,– 6.6, pp. 237-238,

• Clones and Libraries, pp

Page 31: Genetics in the News Recombinant DNA Technology

Polymerase Chain Reaction

…invented by Kary Mullis while cruising in a Honda Civic on Highway 128 from San Francisco to Mendocino,

"It was quiet and something just went, Click!"

Kary B. MullisNobel Laureate, 1993

Chemistry

Page 32: Genetics in the News Recombinant DNA Technology

"THE SUN HAD been hot that day in Mendocino County. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell.

It seemed to be the night of the buckeyes, but something else was stirring.""My little silver Honda's front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back to the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes."

Opening words, Dancing Naked in the Mind Field, © 1998, by Dr. Kary Mullis, Pantheon Books.

Page 33: Genetics in the News Recombinant DNA Technology

Mullis…

... “PCR is a chemical procedure that will make the structures of the molecules of our genes as easy to see as billboards in the desert and as easy to manipulate as Tinkertoys”.

QuickTime™ and aTIFF (LZW) decompressor

are needed to see this picture.

DNA

Page 34: Genetics in the News Recombinant DNA Technology

Making DNA: Components

Cell PCR

ss DNA template

helicase, etc. ?

dNTPs present present

Primer primase ?

DNA Polymerase

DNA

polIII

?

Environment nucleus test tube

Page 35: Genetics in the News Recombinant DNA Technology

Oligonucleotidesspecific primers

...short pieces of synthetic DNA can be manufactured that contain any sequence,

…template specific!

~ Odds of a Specific Sequence

20-mer: 9.1 x 10-13

Page 36: Genetics in the News Recombinant DNA Technology

Making One Strand Of DNA

Add Polymerase

Add dNTPs, etc.

add primer

Page 37: Genetics in the News Recombinant DNA Technology

Making Two More Strands

Must DenatureSeparate Strands

Page 38: Genetics in the News Recombinant DNA Technology

Denaturingcan’t use helicase in vitro

…DNA denaturing conditions such as high heat or low salt concentrations irreversibly denature or inactivate most polymerases,

…dNTPs are not affected by denaturation,

…primers are not affected by denaturation.

Page 39: Genetics in the News Recombinant DNA Technology

Making Two More Strands

Add polymerase, etc.

add primer to second strand

Page 40: Genetics in the News Recombinant DNA Technology

Denaturation Step Bad

…several rounds of in vitro replication could be performed (prior to PCR), however, accumulation of denatured polymerases quickly poisoned the reactions.

Page 41: Genetics in the News Recombinant DNA Technology

…bacteria discovered in a hot spring in Yellowstone Natural Park in 1965,

…lives in salty water that ranges from 70o - 75o C,

…thus, does DNA replication at very high temperatures.

Page 42: Genetics in the News Recombinant DNA Technology

Thermus aquaticus’ Enzymes

…basic research demonstrated that many enzymes isolated from Thermus aquaticus function at very high temperatures,

…temperatures nearing 100o C,

…DNA denaturating temperatures.

Page 43: Genetics in the News Recombinant DNA Technology

Click

…Kary Mullis realized that repetitive rounds of DNA synthesis could be performed by using a heat-stable polymerase,

… Thermus aquaticus: Taq polymerase.

Page 44: Genetics in the News Recombinant DNA Technology

94o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Denature Step~30 seconds

~60o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

Annealing Step~30 seconds

72o

3’--CGTACGTAATACGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

5’--GCATGCATTAT

CTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

5’--GCATGCATTAGGCTACATCGACATCGACTAGCACTG--3’

3’--GCTACGTAATCCGATGTAGCTGTAGCTGATCGTGAC--5’

Synthesis~30 seconds/kb

PCR

Page 45: Genetics in the News Recombinant DNA Technology

Exponential Synthesis

• as few as 1 DNA templates required,

• excess dNTPS,

• excess primers,

• multiple cycles.

Page 46: Genetics in the News Recombinant DNA Technology

Gel Electrophoresis

…electrophoresis is the movement of charged particles in an electric field,

…DNA with it’s phosphate backbone carries a net negative charge,

– DNA migrates toward the positive pole in an electrical field.

Page 47: Genetics in the News Recombinant DNA Technology

Size Resolution

…DNA movement during electophoresis is dependent on several variables,

– strength of the electrical field,

– composition of the matrix (gel),

– charge per unit volume of the molecule,• each nucleotide has roughly equal charge,

• all DNA molecules have the same charge density,

– size.

Page 48: Genetics in the News Recombinant DNA Technology

Agarose Gel

Agarose: a natural polysaccharide derived from agar, a substance found in some algae.

Page 49: Genetics in the News Recombinant DNA Technology

Add DNA to Wells, Apply Charge

DNA moves through gel at a rate roughly equivalent to the inverse log of the number of base pairs.

Page 50: Genetics in the News Recombinant DNA Technology

Visualizing DNA

Ethidium Bromide fluoresces in UV light.

Page 51: Genetics in the News Recombinant DNA Technology

Looks Like This

Page 52: Genetics in the News Recombinant DNA Technology

PCR Applications

…new applications are created every day,

• PCR products can be used for mapping genes,

• PCR products can be used as probes,

• PCR products can be probed,

• PCR can be used to identify genotypes,

• PCR can be used to sequence DNA directly.

Page 53: Genetics in the News Recombinant DNA Technology

Genetics…in the news.

…a hardcopy of the assigned paper will earn you two of the 12.5 points on the quiz.

Page 54: Genetics in the News Recombinant DNA Technology

Syllabus Update

• 6.1, 6.6 - 6.8, 10.1 for Wednesday,

– read the rest of the chapter for review,

– Master the Chapter 6 summary,

– Master 6.8, 6.13, 6.18, 6.19, 10.6, 10.7, 10.9, 10.11,

– Quiz through Chapter 6, 7.1 - 7.5, 10.1 Wednesday.

• Read the Science Paper by Friday.

Page 55: Genetics in the News Recombinant DNA Technology
Page 56: Genetics in the News Recombinant DNA Technology

Molecular ProbingHeterologous Hybridization

…genes (DNA), or gene products (RNA) can be identified based on hybridization to labeled molecules,

…DNA probes are short, single-stranded stretches of nucleic acid that are complementary to target nucleic acids,

– 10 - 1000s of base pairs in length,

…radioactive or fluorescent labeled for detection.

Page 57: Genetics in the News Recombinant DNA Technology

Probe

Add a “labelled” dNTP to an in vitro synthesis reaction.

Page 58: Genetics in the News Recombinant DNA Technology

Southern Blot

Page 59: Genetics in the News Recombinant DNA Technology

Northern BlotmRNA is...

RNA

Page 60: Genetics in the News Recombinant DNA Technology

DNA Libraries

…collections of cloned DNA fragments,

– genomic,

– cDNA (coding sequences).

Page 61: Genetics in the News Recombinant DNA Technology

Genomic Sequences and Coverage

N = ln(1 - P)

ln(1 - f)

N = number of clones

P = probability of recovering a sequence,

f = fraction of the genome of each clone

Page 62: Genetics in the News Recombinant DNA Technology

Genomic Sequences and Coverage

N = ln(1 - .99)

ln(1 - v/2,900,000,000)

v = average vector insert size

plasmid (5000 bp) = 2.7 x 106

phage (20 kb) = 6.7 x 105

BAC (125 kb) = 1.0 x 105

YAC (500 kb) = 27,000 clones

Page 63: Genetics in the News Recombinant DNA Technology

E. coli vs. Humans

# Clones = ln(1 - P)ln(1 - v/g)

P = probability of including any one sequence.

v/g = insert size / genome size

E. coliGenome = 4.6 mbn = 4.6 mb / 20 kb insert = 230P = 0.999

# Clones = 1585

HumanGenome = 2900 mbn = 2900 mb / 20 kb insert = 145,000P = 0.999

# Clones = 1,001,621

Page 64: Genetics in the News Recombinant DNA Technology
Page 65: Genetics in the News Recombinant DNA Technology

cDNA

…DNA synthesized from an mRNA template with the enzyme reverse transcriptase.

Page 66: Genetics in the News Recombinant DNA Technology

Reverse Transcriptase

1. RNA dependent, DNA synthesis.

2. RNA Degradation.

3. DNA dependent, DNA Synthesis.

Error Rate: 1 in 20,000 nucleotides.

Page 67: Genetics in the News Recombinant DNA Technology

mRNA

- polyadenylation.- introns are spliced out.

-AAAA...

Page 68: Genetics in the News Recombinant DNA Technology

cDNA Constructionin vitro

Page 69: Genetics in the News Recombinant DNA Technology

?

cDNA

Page 70: Genetics in the News Recombinant DNA Technology

Genomic vs cDNA

...Jeff’s hemoglobin…

• isolate red blood cells,– red blood cells make lots of hemoglobin, thus the mRNA is

enriched for hemoglobin sequences,

• construct a cDNA library,

• isolation of hemoglobin clones is facilitated,

– genomic: ~1 of 1,000,000 clones,– cDNA from red blood cells: 1 of 3 clones.

Page 71: Genetics in the News Recombinant DNA Technology

cDNA Libraries

…provide a ‘snap-shot’ of the genes expressed in a particular cell, at a particular time, or under specific condition,

…however, do not provide regulatory sequences.

Page 72: Genetics in the News Recombinant DNA Technology
Page 73: Genetics in the News Recombinant DNA Technology

Assignment

• Study figure 6.27, pp 237,

• Be able to describe the steps required to isolate a genomic clone using a cDNA clone as a molecular probe.

Page 74: Genetics in the News Recombinant DNA Technology

Primers?

Page 75: Genetics in the News Recombinant DNA Technology

QuickTime™ and aCinepak decompressor

are needed to see this picture.

Page 76: Genetics in the News Recombinant DNA Technology
Page 77: Genetics in the News Recombinant DNA Technology

Cycle SequencingPCR: 1 Strand Sequencing

• PCR driven DNA sequence procedure,

– non-exponential amplification,

• Dideoxy sequencing method,

– florescent indicators.

Page 78: Genetics in the News Recombinant DNA Technology

Single Strand PCR

Template

dNTPs

1 Primer Taq Polymerase w/ Buffer Cycles

= Polymerization until Taq falls off, linear amplification.

Page 79: Genetics in the News Recombinant DNA Technology

Cycle SequencingChain Termination

Template

ddNTPs

1 Primer Taq Polymerase w/ Buffer Cycles

= Polymerization until Taq hits ddNTP, Taq falls off.

dNTPs

Page 80: Genetics in the News Recombinant DNA Technology

Fluorescent ddNTPs

Plus: a preponderance of dNTPs

Page 81: Genetics in the News Recombinant DNA Technology
Page 82: Genetics in the News Recombinant DNA Technology

Cycle SequencingChain Termination

Template

ddNTPs dNTPs

Lots of each sized fragment are produced, each with a specific florescent base on the end.base

etc.

AnotherTemplate

Page 83: Genetics in the News Recombinant DNA Technology

For Friday!

…READ IT! BRING YOUR COPY!

Page 84: Genetics in the News Recombinant DNA Technology

Exam #1

Increase your study time, or see me to work on study approaches.

year Quiz 1 Quiz 2 Quiz 3 Quiz 4 Exam 1 Exam 2

2007 A 11.5 8 9 7 72%2006 S 12 8 10.7 10 78% 66%2007 S 11.3 9.2 11 11 79% 67%

Mean = 72%Median = 72% Mode = 76%

A range: | | | | | | |B range: | | | | | | |C range: | | | | | | | | | | | |D range: | | | | | | | | | | |Failing: | | | | | | | | | | | | |

Page 85: Genetics in the News Recombinant DNA Technology

Albinism is a recessive trait in humans.Assignment: figure out this

pedigree.14/50