Genomic studies, cloning and trangenic tech

how to identify genes/mutations that are involved in diseases?

look for association of phenotype/ disease w known sequences in genome to identify disease associated regions/genes

gene related AMD

less than 50% of AMD cases can be attributed to mutations in genes

linkage

traits (SNPs, gnees, phenotypes) are linked if they are usually inherited together

• 2 traits that ARE physically CLOSE on a chromosomes have high chance of being inherited together (linked)

• 2 traits on diff chromosomes are NOT linked and have 50/50 chance of being inherited together

single nucleotide polymorphism (SNP)

single nucleotides within the genome that VARY between individuals

Often found outside of genes

used in mapping gene/disease location

why are SNP helpful in mapping disease/gene location?

they may locate near a potential candidate gene that is involved in disease/trait/phenotype under study

recombination

exchange of HOMOLOGOUS segments of chromosomes - mixes up alleles on sister chromosomes (crossover)

• occurs AFTER DNA rep (when chromosomes are condensed and aligned)

• occurs during meiosis (driving force for evolution in euk)

how is family linkage disease be identified?

approach:

• identify proband (1st person who is diagnosed)

• draw pedigree

• isolate DNA from affected and unaffected members

• identify specific SNP alleles present in each subject across the genome (PCR or microarrays)

• identify groups of SNPS that are linked to disease phenotype

• identify nearby genes as candidates for further studies

Genome wide association studeies (GWAS)

• used to detect SNP related diseases in large populations

• uses large numbers of affected and unaffected individuals

benefits for families too small in size for studies or rare/complex diseases, autosomal recessive mutations 

what are the steps in GWAS

1. identify affected pts and unaffected pts (control)

2. isolate DNA

3. analyze SNPs

4. identify SNP that are frequently present in affected pt

5. locate the said SNP

6. identify nearby genes as candidates for further studies

isolation of pt’s DNA

• blood

• cheek swab

• biopsy

• saliva

microarrays

steps:

1. pt sample is isolated, fragmented, and labeled

2. denature sample→ single stranded

3. microarray - allow for hybridization (forming double stranded) - only perfect matches can bond to form double stranded DNA

4. detect flourescence spots (each flourescence spot is an allele A/T/C/G)

5. compare the pattern w a healthy pt to find variance

application of microarrays

• for linkage studies and GWAS (DNA test sample)

• for mutation analysis and genotyping (DNA test sample)

• gene expression analysis (cDNA is test sample)

study of candidate genes

• may be involved in disease process

• test for mutations (PCR, microarray, sequencing)

• mutations should be present in pt DNA and absent in control

• functional analysis of the mutation

• develop animal models of disease (transgenic)

• develop diagnostic tests for detecting mutaiton

• treatments: gene therapy (cloning, crispr)

cloning DNA

• allows isolation and manipulation of specific pieces of FNA in lab

• determine gene structure

• functional analysis

• mutational analysis (test for function of mutated genes)

• use as probes so southern northern

• generate transgenic animals for in vivo studies

• develop gene therapies

cloning DNA and cDNA

• DNA goes thru PCR to amplify specific target sequence, cut w restriction enzymes to make library containing entire genome

• RNA goes thru RT-PCR to amplify single target gene (cDNA), use RT and direct cloning to make library containing enitre transcriptome

• cloning vector (plasmid or virus)

• bacteria to replicate plasmid

steps in cloning

1. cut plasmid (circular DNA that replicate in abcteria, carry antibiotic resistance genes to select bacteria)

2. nsert DNA w restriction enzyme

3. match, anneal, ligate DNA

4. feed the ligated DNA to a bacteria (E. coli)

5. grow bacterial containing plasmid on agar plates

6. antibiotics in agar prevent growth of Ecoli that do not contain plasmid (due to the antibiotic resistance gene in the plasmid)

7. isolate plasmid DNA for future uses

mRNA → cDNA

steps:

1. isolate RNA

2. RT make double stranded cDNA:

• uses Oligo-dT primer binds to poly A tail of mRNA → generate full length cDNA starting 3’ end

3. PCR 

4. Clone PCR product

Gene therapy for RPE65

1. clone the therapeutic gene (RT-CPR to generate cDNA and promoter

2. gene delivery:

• adeno-associated virus (AAV) is used

• virus is infectious → can infect RPE

• virus is replication defective: cannot replicate after infection

• AVV produces, stable, long-term expression

• treatment: subretinal injection to place virus next to RPE

• expression: cells incorporate therapeutic gene and now can make correct RPE65 protein and restore function

transgenic technology

GMO - organisms that has been engineered to contain goreign or modifeied DNA in genome

Random insertion: foreign DNA randomly inserted

KNock-out: deletion of a specific target gene

kock-in: replacement of specfici target w foreign or modified gene

random insertion

1. clone DNA 

2. inject DNA into nucleus of single cell mouse embryo

3. DNA inserts into genome in repeated arrays

4. implant embro into host female

5. breed pups to analyze phenotypes

pros: fast, can generate many animals in weeks

normal gene still intact

can introduce foreign genes

cons: insertion location random

normal gene still intact

may disrupt unknown genes

insertion sites may alter expression of transgene

targeted gene modif by homologous recombination

goal: replace, delete or modify exisiting gene

replace gene w the desired DNA sequence using crossing over during cell division

applications:

make animal models of disease

study gene function and consequences of specific mutations

generate modif in stem cells for therapies

pros and cons of gene modif by homologous recombination

pros:

can replace or modify specific gene in known location

elimiates off target effects

can be condition to allow gene deletion/alteration only in subset of cells or in response to drug

cons:

difficult, expensive, and time consuming

must be done in dividing cells

methods for gene modif by homologous recombination

1. isolate embryonic stem cells (ES cells)

2. clone targeting construct contain desired DNA and antibiotic resistance gene

3. add targeting construct DNA to ES cells

4. cells take up DNA and use homologous recombination during DNA rep tro replace target gene w engineered sequence

5. drug selection to kill any ES cells without new gene

6. screen resistant colonies for recombinants (vs random)

7. inject modified ES cells into multiple blastocyst from normal mouse

8. blastocyst transolanted into female host mouse

9. resulting progeny are chimeras (mixture of cells w normal and modif genes)

10. breed w normal mice and genotype pups using PCR to identify pups carrying modified genome

CRISPR-Cas9

clustered regularly interspaced short palndromic repeats

• developed from bacterial system used to protect against viral infections

• designed to be specific for target gene by virtue of a specific guide DNA sequence

• different strategies used to generate deletions (function loss) or gene edits, or to regulate gene expression

use in: any species, cell type, dividing or non-dividing cells

components of CRISPR

Cas9 - bacterial enzyme makes double stranded cuts in DNA at specific location

Guide RNA (gRNA): single stranded RNA molecule containing predesigned guide RNA sequence complementary to targeted hose sequence and tracrRNA scaffold that interacts w Cas9

donor DNA: required when gene edits, used as template to repair double stranded break in host cell DNA

delivery system/vector: for cloning CRISPR/Cas components and delivering to the target cells, uses plasmid or virus containing cDNA Cas9 gene and template for transcribing guide RNA, donor DNA may be separate or engineerred as part of the same construct

method of CRISPR

1. introduce into cells

2. guide RNA binds to the target site in host DNA genome

3. gRNA positions Cas9 at the target site

4. Cas9 makes a double stranded cut in the host DNA

5. the cell initiates DNA repair:

• without donor DNA: NHEJ generates deletion

• with donor DNA: HDR replaces missing sequence using donor sequence