genetics chapter 13

8/13/2019 Genetics Chapter 13

1/50

Application of Recombinant

DNA Technology

Chapter 13


2/50

Mapping mutations in eukaryotes

Cloning eukaryotic genes

Eukaryotic vectors

Introducing foreign DNA into cells

Mouse genetics - transgenics, knockouts

Human gene therapy

Cloning


3/50

Mapping Mutations in Eukaryotes

DNA Markers

RFLPs(Restriction Fragment Length

Polymorphisms)

VNTRs(Variable Number Tandem Repeats,

minisatellites)

Microsatellites

SNPs(Single-Nucleotide Polymorphisms)


4/50

RFLP

A nucleotide change that results in either eliminationor creation of a restriction enzyme site

technique to detect :

Southern Blot

digest genomic DNA

electrophorese resulting DNA fragments

hybridize using radiolabeled DNA probe thatoverlaps restriction site(s)


5/50

Molecular Characterization of a

RFLP


6/50

RFLPs: Applications

Used to directly diagnose an inherited disease

Sickle cell anemia:

Change in gene sequence of -globin gene

(change of an A to a T in the DNA)

Alters restriction site

Probe hybridizes to DNA region where

restriction site (MstII) is found

In sickle cell anemia, restriction site ismissing due to change in -globin gene

sequence


7/50

RFLPs can distinguish -globin in

wild type and sickle cell anemia


8/50

VNTRs (Minisatellites):

Techniques

Southern Blot:

1. Digest genomic DNA

2. Electrophorese resulting DNAfragments

3. Hybridize using radiolabeled DNA

probe that contains VNTR sequence4. Expose to X-ray film


9/50

Molecular Characterization of a

VNTR

allele A

allele B

locus 1A, 5 copies

locus 1B, 4 copies

locus 3B, 4 copies

locus 2A, 3 copieslocus 3A, 3 copies

locus 2B, 2 copies


10/50

Microsatellites

Tandem Repeats, 2-5 base pairs

(smaller than VNTRs)

Total size of microsatellite:100-1000base pairs

Use PCR to detect using primers that

span tandem repeats


11/50

Advantages of Microsatellites

Used to detect triplet repeat diseases:

Huntingtons disease

Fragile X Used to map genes through

recombination

Scattered throughout genome Large number of alleles in a population


12/50

Molecular Characterization of

Microsatellites


13/50

Microsatellites and Triplet Repeat

Diseases

Age of onset of disease and severity of

disease is related to number of triplet repeats

Huntingtons disease-causesneurodegeneration, due to expansion of

triplet repeats (CAG) in ORF

Fragile X-causes mental impairment, due to

expansion of triplet repeats in front of ORF


14/50

Clinical Diagnosis of Huntingtons

Using Microsatellite Analysis

The higher the number of triplet repeats in the

microsatellite, the more severe the disease

Si l N l tid


15/50

Single-Nucleotide

Polymorphisms (SNPs)

changes in a single nucleotide

SNPs are more randomly and densely

distributed throughout the genome frequency : ~1 out of 1000 base pairs

1.8 million SNPs identified in human

genome

Diff b t SNP d


16/50

Difference between SNPs and

RFLPs

* SNP does not have to be in a site of a restriction endonuclease

M l l Ch t i ti f


17/50

Molecular Characterization of

SNPs by S1 nuclease mapping

Digest DNA into small

fragments

Anneal single-strandedprobe to denatured DNA

Treat with S1 nuclease

(digests single-stranded

DNA)

Electrophorese S1

products

1. If probe and targetDNA sequence aredifferent at SNP site,shortened probebecause S1 cleavesboth strands

2. If probe and targetDNA sequence aresame at SNP site,probe will be full length

SNP

R bi ti M i ith


18/50

Recombination Mapping with

Microsatellites

Follow pattern of inheritance of SNP through

generations on pedigree

Identify SNP that is associated with trait(Which SNP is always seen in individuals with

disease trait?)

Determine which individuals have disease but

do not have SNP associated with disease:

those individuals had recombination event

between the disease gene and the SNP


19/50

Cloning Eukaryotic Genes

Genes associated with a mutant phenotype can be

localized to a chromosomal region byrecombination mapping or by characterizingchromosomal rearrangements (insertions,deletions, translocations), then identifying

mutation and corresponding gene

PositionalGeneCloningidentifying the actualgene based on its location in the genome

Chromosome walking - sequencingoverlapping clones to determine position of gene

Expression patterns to identify candidate genes

Cloning using haplotypemaps

Once general location of mutation in genome is found:

Cl i E k ti G


20/50

Cloning Eukaryotic Genes:

Chromosome Walking

1. Identify molecular markers near gene

2. Generate unique sequence probe

3. Probe genomic library to isolate clones4. Generate restriction map of clones to identifyends of clones that extend the farthest towardgene of interest

6. Use those end sequences as new probeswalk closer to gene of interest using

probe to isolate new clones

Cl i E k ti G


21/50


1. Chromosome Walking

gene may be 100s 1000s of nucleotides

away from markers !

- use unique sequences next to VNTR

and microsatellite markers as probes

if we want to clone a gene we know is mapped

between 2 markers in genome :

generate new probes extending the

furthest in both directions

goal is to isolate a clone

of DNA, between original

markers, that contains the

gene of interest


22/50


2. Identifying Candidate Genes,

Expression Patterns Example: cystic fibrosis (autosomal recessive)

Mapped gene to small region on chromosome 7

containing 4 genes Isolated mRNAs from tissue where cystic fibrosis is

expressed (lungs, pancreas, sweat glands)

Northern blots with probes for 4 genes identifiedonly 1 gene (cystic fibrosis transmembrane

conductance regulator, or CFTR) expressed in all

of the expected tissues

affected patients had mutations in CFTR gene


23/50


Identifying Candidate Genes:

Expression Patterns

CTFR gene identified

(recombination mapping showed it was

between XV-2c and KM-19

Cl i E k ti G


24/50


Identifying Candidate Genes,

Expression Patterns

Cystic fibrosis

Confirmation: clone and sequence CFTRgene from affected and unaffectedindividuals

Affected individuals had mutations in both

copies of CFTR gene

Cloning E kar otic Genes Using


25/50

Cloning Eukaryotic Genes: Using

a Haplotype Map

haplotype : Haploid genotype-specific combinations of markers (SNPs orPCR fragments) on the alleles of a chromosome

for a given individual

within a population, clusters of SNPs do not exhibitrecombination (always found together in genome)



26/50

HapMap (Haplotype Map) project :analyze 1 SNP every 5kb across the entire humangenome in individuals from different geographic populations


a Haplotype Map

Tag SNPs : because of the recombination-free regions, a subsetof SNP alleles can uniquely identify a specific haplotype

- for example, Tag SNPs ATC correspond to haplotype 1

Cl i E k ti G U i


27/50


Association Mapping

association mapping:identifying Tag SNPs that are associated with disease

a gene that may be involved with heart disease is associated

with the Tag SNP in red ; C at this location suggests disease allele


28/50

Eukaryotic Vectors

Yeast vectors

- 2 micron plasmid

Plant vectors

- Ti(tumor inducing) plasmid

Transposable elements

- P elements in Drosophila

Viral vectors

- SV40(Simian virus 40)

can be used to introduce recombinant DNA into

eukaryotic cells, including human cells

Use of Vectors to Express


29/50

Use of Vectors to Express

Foreign Genes

Requires appropriate vector

Requires appropriate promoter elements(so gene is expressed in correct tissue)

Requires appropriate posttranscriptionalprocessing signals

Requires appropriate translational signals

need to express the gene in the correct

cell at the proper time in the proper amount


30/50


Transformation (in prokaryotes)

Treat E. colicells to make them more

permeable to plasmid DNA1. Chemical Transformation - expose

E. colicells to salt (calcium chloride)

2. Electroporation- expose E. colicells toelectrical current

both allow E.colito take in DNA


31/50

Transfection(in eukaryotes)

1. Chemical(calcium phosphate)

2. Electroporation

3. Liposomes

- DNA carried in to cell in membrane

bound vesicles)

4. Injection

5. Biolistic projectiles

- introduce DNA into mitochondria and

chloroplasts on tungsten bullets



32/50

Transfection

Viral vector

Injections

Biolisitics



33/50

Mouse Genetics

Transgenic Micerandom integration of

a foreign gene into the mouse genome

Introduce foreign gene into mouse egg

Implant fertilized egg into female

Analyze genomic DNA in offspring for

transgene

Knockout Mice - physical exchange of

transgene for endogenous gene


34/50

Transgenic Mouse

Inject foreign DNA into male pronucleus of newly

fertilized eggs

T i M


35/50

Transgenic Mouse

Analysis of genomic DNA of transgenic mouse

PCR amplifySouthern Blot

(smaller ;

missing introns)

(2 differently-sized EcoRI

fragments)

Transgenic Mouse: Creating a


36/50

Transgenic Mouse: Creating a

Giant Mouse

Knock Out Mice: Target Vector and


37/50

Knock Out Mice: Target Vector and

Introduction into ES Cells

K k O t Mi


38/50

Knock Out MiceFollow knock out gene in chimeric mice by using

reporter gene (coat color), mate chimeric mice


39/50

Human Gene Therapy

Introduce wild type copy of gene

into patients with defective gene

Severe CombinedImmunodeficiency (SCID)

Absence of adenosine deaminase

results in buildup of deoxyadenosine Toxic to B and T lymphocytes


40/50

SCID Gene Therapy

Insert wild type ADA gene

into retrovirus

Isolate T cells from SCID

patient

Infect T cells with retrovirus

Reintroduce the T cells with

the wild type ADA gene into

patient


41/50

Disadvantages of Gene Therapy

Retrovirus insertion cannot be

controlled

- can insert near protooncogene and

cause T cell leukemia

Can cause immune response to virus

Requires helper virus which can

recombine with retrovirus vector

Human Gene Therapy: Cystic


42/50

Human Gene Therapy: Cystic

Fibrosis

Adenovirus as a vector

Infects lung epithelial cells

Does not integrate into hostchromosome

Maintained as extrachromosomal DNA

Requires continual application forpatients


43/50

Cloned Organisms

Genetically identical

Replace nucleus of egg with nucleus

from epithelial cell Mitochondrial genes still remain from

host cell

Large number of nuclear transfersrequired (over 1000)


44/50

Cloned OrganismsDolly (1997)

Somatic nucleus

donor and Snuppy

(2005)

Snuppy

and

surrogatemom


45/50

Disadvantages of Cloned Organisms

Require multiple nuclear transplants

Cloned animals have shorter life spans

Cloned animals have more propensityfor disease and physical abnormalities

- at age 6 Dolly had lung cancer andsevere arthritis

Potential disruption of gene functioning

Applications of Genetic Engineering


46/50

Applications of Genetic Engineering

Medicine

Basic knowledge of how genes work

Identifying genes that cause diseases

Producing large amounts of proteins and

antibodies to help fight disease Gene therapy

Transgenic animals : potential for diseasemodels

Cloned animals : potential to produce organsfor human transplants

Applications of Gene Cloning


47/50

Applications of Gene Cloning

Agriculture

Transgenic cropsresistant to pests

resistant to frost

resistant to premature ripeningresistant to herbicides

Industry

Engineering bacteria to break down toxic

wasteYeast that can convert glucose to ethyl

alcohol to replace fossil fuels

C


48/50

Ethical Considerations

Genetically modified organism (GMO)(plants)

Are they safe for human and animal

consumption? Is it ethical to plant GMOs which

encourage the use of herbicides?

Can we control the spread of GMOsonce they are planted?

Ethi l C id ti


49/50

Ethical Considerations

Cloning Organisms and Individuals

PreimplantationGeneticDiagnosis(PGD) toreduce likelihood of bearing child with geneticdisease

- in vitro fertilization

- remove 1 cell from embryo at 8-cell stage,

screen it for mutation or desired trait

- implant 7-cell embryo in mothers uterus if

desired trait is present (or undesired is absent)

Is test dangerous to individuals later in life?

Is manipulation of a childs genome ethical?

Preimplantation Genetic Diagnosis


50/50

Preimplantation Genetic Diagnosis

genetics chapter 13

Documents