Chapter 5 Powerpoint 2008 - University of Arizonacbc.arizona.edu/classes/bioc461/Chapter5Powerpoint.pdf · POLYMERASE CHAIN REACTION (PCR) for gene detection and amplification. Sac

HOUR EXAM 1: September 29, 2009 (Tuesday)

EXAM WILL COVER:

CHAPTER 25

CHAPER 4

CHAPTER 5 TO END of Sept 24 Lecture

EXAM 1 REVIEW: Monday, Sept. 28, 2008, 5-6:00 PM, BSW208

CHAPTER 5

Exploring Genes and Genomes

Sept 18, 2008

Dangers of Genetic Engineering?

The Bubble Boy

Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease. (2000)

[Bubble boy disease] Cured by introducing engineered DNA coding for cytokine receptor γ-chain [bone-marrow transplant]

Problems with X-SCID gene therapy will be discussed near end of Chapter 5.

CHAPTER 5: EXPLORING GENES and GENOMES

LECTURE TOPICS (4 Groups)

1) RESTRICTION ENZYMES CUT DNA

2) GEL ELECTROPHORESIS OF DNA

3) DNA SEQUENCING, RNA SEQUENCING, DNA SYNTHESIS

4) POLYMERASE CHAIN REACTION (PCR)

5) RECOMBINANT DNA CONSTRUCTION AND GENE CLONING

6) DNA CLONING VECTORS

7) GENE LIBRARIES: MAKING AND SCREENING THEM

8) CHROMOSOME MAPPING

9) EXPRESSION OF CLONED GENES

10) ENGINEERING NOVEL PROTEINS

Recombinant DNA technology(started in mid-late 1970's)

• An incredibly powerful set of tools for gene manipulation.

• Methods associated with this "technology" make genetic

engineering a reality.

• DNA (genes), RNA, and protein structure and function can be

altered by design for beneficial (or detrimental – biological

warfare/terrorism?) results.

KEY TOOLS and METHODS OF GENE EXPLORATION

1. ENZYMES to cut, join and replicate DNA in test tubes (in vitro)

a) restriction enzymes are DNA cutters

b) DNA ligases are DNA joiners

c) DNA polymerases for DNA replication

2. GEL ELECTROPHORESIS to separate and isolate specific DNAs

3. BLOTTING METHODS based on hybridization (BASE-PAIRING)of complementary DNA and/or RNA

4. SOLID PHASE methods to sequence and synthesize DNA

5. POLYMERASE CHAIN REACTION (PCR) for gene detection andamplification

Sac II Restriction Enzyme Recognition Site

180 degree rotation(2-fold axis of symmetry

Ex: Eco RI

G AATT C

C TTAA G

G AATT C

C TTAA G3ÒH 5` P

5`P 3` OH

Cut

Cut

5'

3'

3'

3'

3'5'

5'

5'

single strand overhang

Blunt ends

4 bp site

6 bp site

Symmetry axis

Cut

DNA cut with three different restriction enzymes

Longer DNA

DNA moves to

(+)electrode

Shorter DNA

Detect fluorescence of a dye (Ethidium Bromide) that binds to DNA

(-)

(+)

(-)Agarose gel

electrophoresis of DNA

Polyacrylamide Gel Electrophoresis for DNA Sequencing

(-)

(+)

Longer

Shorter

ELECTROPHORESIS

ALL adjacent

bands differ in length by only ONE

base

DNA Blotting: “Southern blot” method devised in 1975At First rejected, Ed Southern’s paper has since been cited about 50,000 times.

Southern found that:1) DNA transfers by diffusion from the gel to the Millipore (nitrocellulose) filter. 2) DNA on filter can be denatured and detected by hybridizing to a radioactive probe.

[“Probe” is a DNA or RNA that is identical or related enough to form stablebase- paired regions – i.e, has complementary sequences to the DNA.]

DNA

DNA Blotting and Hybridization to a “probe”

“Probe” is either identical or related

complementaryDNA (or RNA)

(X-Ray film)

1.1 kb1.3 kb

Normal Sickle cell

Restriction fragment length polymorphism (RFLP):

Ex: MstII RFLP for Sickle-Cell detection

Normal Sickle-CellParent CarriersParent carriers

*

CHAPTER 5: EXPLORING GENES

LECTURE TOPICS









75 bases tRNA - (1964) [slow, complicated method]

5386 bases NX174 DNA (1977) [fast]

155,844 bases tobacco chloroplast DNA (1986)

1.8 million bases H. influenzae (1995)

3 million bases E. coli (1997)

3 billion bases human (2000!!) [fastest!!!!]

DNA Sequencing: Landmark genomes completed

Haemophilus influenzae genome (1995)

1.8x106 bp

Year 2000

Human DNA sequence

completed!! (sort of…. mostly)

(3x109 bp)

Gene sequences are now precisely located on human chromosomes

Gene sequencing shows how similar chromosomal location of related genes are in different organisms

My First DNA SequenceThe same tech megatrends that are reshaping grown-up gadgets are revolutionizing kids' toys. Nowadays, youngsters can race nitro-powered remote control trucks, fiddle with programmable robots, and guest-star in the latest sitcoms. If those aren't sophisticated enough for your brainiac tykes, the Discovery Kids DNA Explorer helps junior scientists extract and map real deoxyribonucleic acid. As third-grade science projects go, this is light-years beyond the ol' baking soda volcano. Next step: cloning Fido.[ DNA Explorer (ages 10 and up): $80, http://www.discovery.com/ ]

DNA sequencing by chain termination of DNA synthesis method: (Sanger method)

Reaction Conditions that must be satisified:• Reactions specific for each base (A,T,G,C)

• Controlled random reactions on all elongating DNA chains

• Equimolar collection of reaction products (same frequency of DNA chain stopping for all 4 bases and all elongating chains)

Chain terminating dideoxynucleotide

DNA sequencing by chain termination of DNA synthesis

DNA sequencing by Chain Termination

Reaction products For A :ddA

ddA

ddA

ddA

(etc.,etc. for as many T’s as there are in template)

****5` 3`

• Do same for C, G, T

• Denature and separate by electrophoresis

• Detect fluorescence

Example: for A[Template]

[Primer]

dNTP’s too*

DNA Sequencing Movie

Dideoxy_Sequencing_of_DNA.mov[From Berg et al. textbook website]

DNA Sequencing Gel (from Lehninger)

(+)32P, 35S

radioactivity

Longer (3` end of new chain)

Shorter (5` end, nearer primer)

Electrophoresis(-)

Now use fluorescence

l l l lA C G T

A C G T

DNA Sequencing: Automated Detection by Fluorescence• Key to human genome project

• Read 400-1000 (even more!) bases at a time. [1 x 106bases/day/machine].

• All 4 dNTP reactions run in same tube at the same time with 4 fluorescent ddNTPs

• All 4 detected at same time.A T G C

Shorter(near 5ènd of primer)

(Time) Longer(3ènd of growing chain)

CHEMICAL SYNTHESIS OF DNA (SOLID PHASE, AUTOMATED METHODS)

Make oligodeoxynucleotide chain from 3’ to 5’ end (short DNA, parts of genes for probes and primers) [oligo means 2-20 bases]

Use in DNA/RNA sequencing, cloning, and gene probing by hybridization

Easy to make DNA 100 nucleotides long (18-20 used most often)

Chemically synthesized DNAs are key to protein engineering by site-directed mutagenesis.

Start synthesis with blocked nucleotide linked to a solid support (glass bead).

Blocked nucleotide for DNA Synthesis:Phosphite triester method

5`-block

protected by NH2 groups(A,G,C,T)

Modified 3`-end

Solid Phase DNA Synthesis

v

5` 3` DMT off

5`

Last steps: 1. Remove βCE2. “deprotect” bases NH3 removes all

3`


LECTURE TOPICS

3) DNA SYNTHESIS – LARGE ARRAYS OF DNAs








Synthesizing Oligonucleotides Movie

SynthesizingOligonucleotide.mov[From Berg et al. textbook website]

A DNA chip

DNA Chip: 256 Octanucleotides• Ink jet printer head robots deposit DNA on chips

• Hybridize with 8-mer test sequenceGCGGCGGC

All Octanucleotides (8 bases): 48 = 65,536

Need chip of 1.6 cm2 with 50 μm sites

Only 32 steps of synthesis (8 hours)

DNA chips to identify BCRA1 mutants (breast cancer gene)

C T

Use a DNA chip with 48,300 spots

Green: normal

Red: test gene (mutant)

Yellow: red and green superimposed (red is mutation)

DNA chip study of gene expression in 84 human breast tumor samples.

Red: Genes with higher than normal activity in tumor.

Green: genes with lower than normal activity in tumor.

DNA chips identify changes in yeast gene expression under different conditions.

[Red: higher than normal]

[Green: lower than normal]

1918 pandemic Flu virus induces altered mouse gene expression:

Studied by DNA chip analysis.

Data gives clues about what may be gene targets in humans of H5N1 bird flu

Modern human flu strain

1918 pandemic flu strain

(overexpressed)

Connectivity Concept: Using gene expression profiles (DNA chips) to connect perturbed gene expression (like in diseases) with “correcting” drug treatments. [Lamb, et al, Science 313, Sept. 29, 2006 p.1929]


LECTURE TOPICS

2) GEL ELECTROPHORESIS OF DNA (continued)









DNA polymerase chain reaction: Concept(starting with only one of two strands of DNA)

3` 5`

5`3`

3` 5`

5`3`

3` 5`

5`

5`

3` 5`

3`

3`

3` 5`

5`3`

3` 5`

5`3`

+

+

Original DNA (n=0)

1 Copy(n=1)

2 Copies(n=2)

primer1

primer1

primer2

(Taq) DNA polymerase + dNTPs

Denature (separate strands), then cool mixture

template

Heat

Polymerase Chain Reaction (PCR): Concept

Many cycles DNA Amplification

1 Copy (D.S.)

One “cycle”(n=1)

2 copies (D.S.)

&Primers

Short products predominate. They are amplified as 2n

cycle 1

cycle 2

cycle 3

n cycles

Short products (target sequence)

Polymerase Chain Reaction Movie

Polymerase_Chain_Reaction.mov[From Berg et al. textbook website]

PCR: # Copies of DNA = 2n

1

2

48 16 32 64 Copies

Cycle Copies

0 1

1 21

2 22

3 23

4 24

5 25

6 26

n 27

Start with one copy

(D.S.)

After 45 cycles: 245 = 3.5 x 1013 = 3,500 billion copies

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This official forensic kit contains realistic experiments that allow your child to solve a mystery in the style of the investigators on the popular television series.

For ages 10 and up.

USE OF PCR in CRIMINOLOGY

(D) (D)D = Defendant

V = Victim

* Data shows that defendant’s clothes have victim’s DNA (in bloodstains)

* * *

Probability of a random DNA match is 1 in 33 billion!!


LECTURE TOPICS










CONSTRUCTION, CLONING AND EXPRESSION OF DNA

Novel combinations of genes can be cloned, amplified and expressed in foreign environments

KEY STEPS / PROCEDURES

1. Construct recombinant DNA molecule.

• DNA inserted in a vector

2. Clone and amplify DNA in vector`s host cells

3. Select individuals that have recombinant gene

• ***Antibiotic resistance***

• Gene probing

• Antibody reaction

Foreign DNA (insert)Plasmid vector

[cut both with same enzyme]

[Join with DNA ligase]

Construct recombinant DNA molecule

Select cells that have recombinant DNA

Cloning

CLONING DNA

Introduce DNA into host cells by transformation or viral infection

“Cut”

“Join”

Cutting and joining DNA molecules

*

Linkers- to clone blunt-ended DNA

Synthetic Linker DNA

Step 1: “Join”

Step 2

Clone

Plasmid Cloning Movie

Plasmid_Cloning.mov[From Berg et al. textbook website]

All Recombinant DNA Vectors:

Properties must allow to:

1. Clone in specific sites

2. Select [antibiotic, β-galactosidase]

• Insertional inactivation

3. Replicate in host cells

12

2

2

3

1

Ex: Prokaryotic (E.coli) Plasmid Vector: Example that satisfies requirements

1 Select

2 Specific cloning sites

3 Replication

pUC18 prokaryotic plasmid vector: Polylinker with many cloning sites

1

1

2

3

1 Select

2 Specific cloning sites

3 Replication

(An E.coli vector)

SOME CLONING VECTORS

• Plasmids (Autonomously replicating)

• - [2-6 kbp inserts – prokaryotic gene sizes]

• - Insertional inactivation of antibiotic resistance orβ-galactosidase activity

• Lambda phages (good for libraries of eukaryotic cDNA’s) [10-23 kbp inserts – a few eukaryotic genes]

• YAC’s (yeast artificial chromosomes) – For big pieces of chromosomes[1 Mb inserts (106 base pairs; about 1000 genes)]

• Ti plasmid of Agrobacterium tumefaciens (for plant genetic engineering [one or a few genes insert into chromosomes]

Bacteriophage lambda (λ) lifecycle

*

Bacteriophage lambda (λ) as a cloning vector

L R

Delete this piece

L R

Recombinant λ DNA in new phage particle

L R

Yeast Artificial Chromosome (YAC)

Up to 106 bp

To clone large chromosomal

pieces

PLANT GENETIC ENGINEERING(With Ti plasmid of Agrobacterium tumefaciens)

Crown Gall DiseaseCaused by

Agrobacterium

Cells are transformed

T-DNA inserts in chromosomesDelete and

insert genes

Delete these genes

Ti plasmid

T-DNA

Delete some T-DNA

*

Infect with Agrobacteriumcontaining recombinant Ti plasmid that has a selectable marker

Agar with growth hormones and kanamycin (antibiotic for selection)

Kanamycin-resistantplants contain the foreign gene

Transformation and selection of transgenic plants


LECTURE TOPICS










MAKING GENE LIBRARIES

Genomic library – from all fragments of a cell`schromosomal DNA (large pieces)

• YAC’s

• Bacteriophage lambda

cDNA library – DNA copies of a mix of all cellular mRNA (a few kbp cDNA’s of all expressed genes)

• lambda phage

• plasmids

λ Genomic Library Construction

1. Cut

2. Ligate3. Get recombinant λ DNA

4. reconstituted λ virus

5. Get more virus from infected cells

L R

*

Making a cDNA library from mRNA

5`-------CCnCCPrimer

[OH-] cuts RNA

Primer

2nd strand cDNA

1st strand

Add linkers Clone in a vector

Terminal transferaseadds dG’s

SCREENING GENE LIBRARIES(searching for a needle in a phagestack!!)

Need to screen 500,000 clones to find a specific sequence in a genomic library

• Easier for “abundant RNA” in a cDNA library

• Immunochemical (antibody) screening of an expression library

• N.A. hybridization screening (with gene probe)

• Make synthetic DNA probes (can even predict DNA sequence by reverse translation of protein)

• Chromosome walking (connecting long pieces)

• map whole chromosomes (Clone in λ, YAC, or BAC)

Screening gene (or cDNA) libraries: replica plating

1) λ phage plaques

or

2) bacterial colonies

Hybridize to DNA probeor

Antibody reaction Clone containing

gene

Master plate Autoradiogram of replica plate

DNA/DNA hybrid or antibody

Make replica plateon filter

Screening a lambda library (genomic or cDNA)

(or cDNA)

Get recombinant λ DNA

Infect cells Isolate individual plaques

Screening a lambda genomic library: use DNA probe

Individual plaques

(dead cells –clear spots)

Hybridize to gene

DNA probe λ clone containing

gene

Master plate Autoradiogram of replica plate

DNA/DNA hybrid

Make replica plate

Immunological Screening

Antibody detects cells that contain the protein of interest

“Blotting” replica

*

*

*

Bacterial colony or λ

plaque

For cDNA library(protein expressed)

1.

2.

Reverse translation: To find DNA clone coding for a specific protein

256 possible DNA coding sequences (2x4x2x2x4x2 = 256)

1. Use amino acid sequence to predict gene sequence.

2. Synthesize (and label with radioactivity) 21bp DNA of all possible gene sequences and use to “screen” a library. (need to consider degenerate codons)

Screening OligonucleotidesMovie

Screening_Oligonucleotide.mov[From Berg et al. textbook website]

Ways to use recombinant DNA Technology

From Gene to unknown protein

From Protein to get unknown Gene


LECTURE TOPICS










“Chromosome Walking” (use YACs)

Start with DNA (A’): hybridize to library

Order of A D deduced by successive hybridizations


LECTURE TOPICS



Dangers of Genetic Engineering?

Be careful what you ask for!

DNA vector delivery

Microinjection

Gene gun

viruses DNAelectroporation

liposomes

Delivering DNA vector to cells

• Calcium phosphate precipitated DNA

• Microinjection

• Virus vectors (SV40, vaccinia, retroviruses)

• GENE GUN (microprojectiles coated with DNA)

• Liposomes (coat DNA with “cell membrane”)

• Electroporation

Electroporation: DNA delivery to

plant cellsMake holes in cell wall

Protoplast

CLONED GENES / VECTOR SYSTEMS:(some examples)

• Human Proinsulin cDNA

• Engineered mammals (giant mice – somatotropin gene)

• Engineered Plants (Ti-plasmid of Agrobacterium tumefaciens) Ex: Flavr-Savr tomatoes (Calgene)

• 1918 pandemic influenza virus – reconstructed from cloned pieces of its genome. [samples recovered from victims buried in Arctic permafrost]

Human insulin cDNA

CLONING Expression

(No introns)

Identify proinsulin by antibody reaction

Creating a Transgenic Mouse

Creating_a_Transgenic.mov[From Berg et al. textbook website]

Engineering a Giant Mouse:Somatostatin gene injected into mouse male pronucleus

Engineered Giant Mouse: Somatostatin

Add Cd++

• 2x normal weight

• 500x hormone levels

Control: no Cd++ or

no extra rat growth hormone

(somatostatin)

Somatostatin

Microinject plasmid

PLANT GENETIC ENGINEERING(With Ti plasmid of Agrobacterium tumefaciens)

Crown Gall DiseaseCaused by

Agrobacterium

Cells are transformed

T-DNA inserts in chromosomes

Delete these genes

Ti plasmid

T-DNA

Delete some T-DNA

*

Delete and insert genes

FLAVR SAVR Tomatoes: FDA APPROVED FOR SALE

CALGENE: to market in 1995

“Antisense”Gene

Disruption Strategy

Gene is transcribed as complementary RNA to mRNA for normal plant gene involved in fruit ripening.

from the New Yorker, 1994

Ti plasmid, mouse vector chromosome insertions: relate to concept of Homologous/Non- homologous Recombination. These events result in:

• Gene replacement, Gene disruption, or random chromosome insertions

The Bubble Boy

Gene Therapy of Human Severe Combined Immunodeficiency (SCID)-X1 Disease. (2000)

[Bubble boy disease] Cured by introducing engineered DNA coding for cytokine receptor γ-chain [bone-marrow transplant]

Correction of ADA-SCID by Stem Cell Gene Therapy Combined with Nonmyeloablative Conditioning (2002)

Cured by introducing engineered DNA coding for the enzymeADA (Adenosine deaminase)

BUT: Random insertions correlated with leukemia occurrence after a few years in treated X-SCID patients.

• leukemia correlates with insertion of DNA into a gene.

• Gene disruption - loss of normal function leads to leukemia.


LECTURE TOPICS

9) EXPRESSION OF CLONED GENESRNAi gene silencing


RNA interference (siRNAs)

A mechanism for gene disruption.

RISC: RNA-induced gene silencing complex

[21 bp dsRNA]

ssRNA

Gene Silencing disrupts gene expression.

**2006 Nobel Prize in Medicine!!

Practical Applications of Cloning by Recombinant DNA Methods.

NOT a DINOSAUR, BUT: [Fall, 2005 news]An infectious flu virus was created in the lab

of from cloned pieces of its genes.

• Influenza virus that caused 1918 Spanish flu pandemic. (killed 20 million people.)

• Used “Jurassic Park” type DNA cloning methods– RNA cloning (cDNA’s) in a DNA vector.

• 1918 virus is a bird flu strain. – That’s why H5N1 bird flu is so worrisome.

1918 pandemic Flu virus induces altered mouse gene expression:

Studied by DNA chip analysis.

Modern human flu strain

1918 pandemic flu

strain

PROTEIN ENGINEERING by SITE-SPECIFIC MUTAGENESIS

PROTEIN ENGINEERING:

SITE-SPECIFIC MUTAGENESIS

Modify coding information to get a different amino acid sequence

Change base with mismatch primer for DNA replication

SITE-SPECIFIC MUTAGENESIS

A mismatch changes only one codon

How to do it??

1. Hybridize mismatch primer

2. Replicate DNA (plasmid)

3. Half of daughter DNA has mutation!!

In Vitro Mutagenesis Movie

In_Vitro_Mutagenesis.mov[From Berg et al. textbook website]

Current and Future Applications ofRECOMBINANT DNA TECHNOLOGY

• Complete chromosome gene maps

• Whole genome sequencing by shotgun approach

• Discovery of molecular bases of development, evolutionary relationships

• New proteins with new functions (or old proteins with new functions!)

• Human hormone synthesis in bacteria

• Antiviral agents

• AIDS vaccine development

• New pharmacological agents (proteins, RNA, DNA)• Antisense RNA therapy (RNAi, gene silencing)

• Medical diagnostic reagents (gene probes) for detection of genetic diseases, infections and cancers

• Gene therapy: delivery with disarmed viruses to alleviate diseases caused by known gene defects.

• Agricultural revolution with animals having altered traits, more nutritious plants, heat/drought resistant crops, etc.

• Forensics - molecular detectives

Current and Future Applications of RECOMBINANT DNA TECHNOLOGY

Documents

Chapter 5 Powerpoint 2008 - University of Arizonacbc.arizona.edu/classes/bioc461/Chapter5Powerpoint.pdf · POLYMERASE CHAIN REACTION (PCR) for gene detection and amplification. Sac