1 molecular technologies discovery of dna structure/function (1953) molecular biology powerful...

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Molecular Technologies

Discovery of DNA structure/function (1953)

Molecular Biology

Powerful Techniques - Gene Analysis & Manipulation

Widely Applicable

Ecology, Behavior, Medicine, Agriculture

Pharmaceuticals, Horticulture, Animal Breeding, etc....

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Denaturation of DNA

High temperature, high pH - break H bonds

hyperchromatic effect

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Denaturation of DNA

Relative G:C content

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Renaturation of DNA

Homology affects renaturaton (hybridization)

Stringency [Salt] Temperature

High Low High

Low High Low

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Renaturation of DNA

Concentration affects rate of hybridization

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Renaturation of DNA

Concentration affects rate of hybridization

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Nucleic Acid Probes

DNA/RNA probes

Target sequences

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Phylogenetic Relationships

Amount bound ~ homology

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In situ Hybridization

FISH

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In situ Hybridization

Chromosome Painting

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In situ Hybridization

Detecting Chromosomal Rearrangements

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One specific sequence at each spot

Hybridize with labeled RNAfrom different cells or individuals (red or green)

Detect binding and interpret results

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Microarrays (Gene Chips)

Demonstration: http://www.bio.davidson.edu/Courses/genomics/chip/chip.html http://learn.genetics.utah.edu/content/labs/microarray/

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Microarrays (Gene Chips)

mRNA isolated from cells (cancer/normal; aerobic/anaerobic)

Converted to cDNA Fluorescent labels

http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

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Microarrays (Gene Chips)

Mixture of labeled cDNA hybridized to microarray

Specific binding Scan and evaluate expression

http://www.bio.davidson.edu/Courses/genomics/chip/chip.html

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Microarrays (Gene Chips)

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Microarrays (Gene Chips)

Applications:

Evaluate gene expressionDevelopment, Cancer, Disease progression

Determine presence of specific mutations Genotyping, SNP association studies

Detect infectious genomes

Virus, Bacteria, etc.

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Restriction Endonucleases

Bacterial immune system

Recognize and cleave foreign DNA

Names

EcoRI - E. coli strain RI

HinDIII - H. influenzae strain DIII

Each recognizes and cuts a specific sequence of DNA

Specific molecular scissors

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Restriction Endonucleases

Palindromic Sequence

‘AND MADAM DNA’

5’>3’ = 3’>5’

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Restriction Endonucleases

Ends:

Blunt

or

Sticky

(complementary)

3’ or 5’ overhangs

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Restriction Endonucleases

Recognition Methylation

Enzyme Site Sensitive ?

AluI 5’...AG CT...3’

No3’...TC GA...5’

BamHI 5’...G GATCC...3’

No3’...CCTAG G...5’

BspEI 5’...T CCGGA...3’

Yes 3’...AGGCC T...5’

KpnI 5’...GGTAC C...3’

No3’...C CATGG...5’

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Recombinant DNA

RE fragments from two sources joined by DNA ligase

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Cloning DNA

DNA inserted into vector for growth in another cell

Vector enables growth in host cell (ori or ARS)

Selective markers (antibiotic resistance, nutritional requirement)

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Plasmid Vector

Bacterial Vector

pUC19

pBluescript

LacZ at MCS

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Eukaryotic Vectors

Yeast Artificial Chromosome

Shuttle Vectors

CEN; ARS plus ORI; markers

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Gene Cloning

Source of DNA Insert

Genomic DNA - dsDNA - coding and noncoding

cDNA - RNA > DNA - coding only

One specific gene

or

Comprehensive collection -

Genomic

Library, cDNA Library

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Genomic Library

Clone collection of

overlapping fragments (contigs)

Ave. fragment size

256 bp = (44)

4096 bp = (46)

Comprehensive library

for 3 x 109 bp genome

4096 bp fragments

> 732,000 clones

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Genomic Library

Probability of having at least one copy of any DNA

sequence in a genomic library

N = ln(1-P) N = number of clones needed ln(1-f) P = probability desired

f = ave. size fragment

cloned/genome size

Ex. Human genome 106 kb; if fragments average 15 kb,

More than 920,000 clones are needed

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cDNA Library (Expression Library)

cDNA - complementary DNA

copy mRNA - reverse transcriptase, polydT primer

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Transferring Recombinant DNA into Host Cells

Transformation

Each colony contains vector, Only some have insert

Clones - each with specific segment of genome

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Identifying Transformants Containing Gene Inserts

Blue/White Screen - pBluescript

Ampicillin resistant

lacZ gene-galactosidaseIPTGXgal (white) > blue

White coloniesall contain insert in MCS

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Screening Clones for Specific Gene Sequences

Detection of specific sequence - probe

Colonies transferred to membraneDNA released, hybridization with probe

Identify clone with desired gene

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Screening cDNA Libraries for Specific Gene Products

Detection of protein produced by cells with desired gene

Identify clone expressing gene

Specific Antibody

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Screening Clones for Ability to Rescue Mutants

DNA genomic library in shuttle vectorused to transform eukaryotic cells with defective gene

Only those with good copy of the gene grow

Permissive (30OC) Restrictive (37OC)

Complementation

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Screening Clones for Ability to Rescue Mutants

Cloning of yeast ARG1 gene

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Restriction Mapping

Relative positions of RE sites along DNA - molecular markers

670 1,500 500 250

BstEB

bp distances

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Restriction Mapping

Specificity of enzymes - reproducible fragments produced

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Determining Sizes of Restriction Fragments

Gel Electrophoresis - Agarose (200 bp+), Polyacrylamide

Determine distance migrated *Extrapolate size

*

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Determining Sizes of Restriction Fragments

Visualize DNA bands - Ethidium Bromide, UV transilluminator

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Generating Restriction Maps

Complete Digestion with HindIII and/or EcoRI

kb

kbkb

kb

kb

Answer:

4 H 7 E 9

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Generating Restriction Maps

Partial Digestion with PstI

Possible partial digestion fragments (*complete)

19, 15, 14, 12, 10, 8*, 7, 5*, 4*, 2* kb

Is a 6 kb fragment possible? A 13 kb fragment?

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Generating Restriction Maps

Partial Digestion with SalI

Which fragmentsare adjacent?

4.2 = 3.5 = 8.2 =

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Using Restriction Maps to Analyze Constructs

Characterize insert in recombinant DNA of clone

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Using Restriction Maps to Analyze Constructs

Orientation of insert can be determined

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Analysis of Genomic Sequences

Human genome - cut with RE (6 bp site) - ~7.3 X 105 fragments

How can you find and identifya specific fragment?

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Southern Blot Analysis

Southern Blot

DNA transfer -

detect fragment

specific probe

Northern BlotRNA

Western BlotProtein

Transfer tonylon membrane

Hybridize with probe

Visualizeautoradiographychemiluminescence

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RFLP Analysis

Restriction Fragment Length Polymorphisms (RFLP)

Basis for polymorphic DNA sequences

1. Change in RE cleavage site

Gel Pattern

A

a

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RFLP Analysis

Basis for polymorphic DNA sequences

2. Change in number of repeats

VNTR (variable numbers tandem repeats: 10 - 1000

bp)

STRP (simple tandem repeats: CAGCAGCAG)

Southern blot with probe to repeat

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SNP Analysis using Southern Blot

Single Nucleotide Polymorphisms that affect restriction sites

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Mutation Detection using Southern Blot

Detection of Sickle-cell gene by DdeI fragment

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Association of RFLP with Disease Allele

If RFLP pattern and disease are inherited together,

the RFLP site (probe) and the disease gene are linked.

If < 1% recombination, they are within 1 million bp of each other.

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Association of RFLP with Disease Allele

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DNA Fingerprinting

Alex Jeffreys - Southern Blot, probes to hypervariable regions

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DNA Fingerprinting

Paternity test Forensic

test

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Polymerase Chain Reaction - PCR

Amplification of specific region of DNA - specific primers

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Polymerase Chain Reaction - PCR

Thermal cycler - 25+ cycles - 3.4 x 107 copies of template

95OC 50 - 60OC 72OCdenature anneal extension

Major productvisible on gel

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Polymerase Chain Reaction - PCR

Using PCR to determine which STR alleles are present

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Polymerase Chain Reaction - PCR

Using PCR to study SNPs that affect RE sites

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DNA Profiling using PCR

Different primer set for each allele used in multiplex PCR

Products size-separated by capillary gel electrophoresis, HPLC

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DNA Profiling for FBI

STR profile -

13 STR loci,

X/Y markers,

2 other loci

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Determining Probability of Match in DNA Profiling

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DNA Sequencing

DNA template - labeled primer (or nucleotides), DNA polymerase, dNTPs, dideoxynucleotides (ddNTP)

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DNA Sequencing

Polyacrylamide gel electrophoresis, autoradiography

Sequence deduced from bands on autoradiogram

5’ A- G-C-C-T-A-G-A-C-T 3’

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DNA Sequencing

Capillary gel electrophoresis, fluorescent ddNTPs

http://www.wellcome.ac.uk/News/2009/Features/WTX056032.htm

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Gene Therapy

Somatic Cell Therapy - feasible for single gene disorders

Somatic cells isolated from individual with defect

Transformed with cloned wild-type gene

virus, direct injection

Cells reintroduced into individual

bone marrow, blood stream

Ideally, cells survive and make gene product

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Gene Therapy

Transgenic cell - contains artificially introduced gene

Transgene - gene used

Examples of successful gene therapy

SCID (severe combined immunodeficiency) adenosine deaminase (ADA)

periodic infusions of transformed T cells

Sickle-cell anemia - bone marrow - permanent cure

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Biotechnology - Transgenic Animals

Expression in mammary tissue - -Lactoglobulin promoter

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Biotechnology - Transgenic Animals

GloFish - Available at That Fish Place

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Biotechnology - Transgenic Plants

Agrobacterium tumefaciens - Ti plasmid

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Biotechnology - Transgenic Plants

Roundup™ Readytobacco

Modified bacterialgene for EPSPS

- herbicide resistance

(5-enolpyruvylshikimate-3-phosphate synthase)

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Applications of Genetic Technology

Isolation and characterization of genes and genomes

Potential for gene therapy and molecular diagnosis

Agriculture: plants - insect resistance, enhanced nutrition/flavor animals - increased production

Environmental: oil-eating bacteria

Manufacture of human gene products:human insulin, growth hormone, clotting factor, etc.

Genome projects - tremendous potential for rapid development