exam 3: distinguish individual genomes Flashcards
why would u want to distinguish individual genomes
*human disease research
-ID what genes cause disease and how mutations affect them (determine if an individual is suspectible)
-disease prevention (minimize expressivity)
–> embryo pre-implantation genotyping
—> preemptive therapy
*ID
-ID the dead
-ID crime suspects
-ID parents/ relatives
*family linage
*wildlife forensics
ethical issues to who should have access to information and how should they use it?
-preemptive therapy
if u know u carry specific disease-causing allele (pre-existing condition), youcould take steps to prevent onset of disease before symptoms develop, BUT ur insurance company may drop your coverage if they know u have the allele
polymorphisms
> 1 allele at a specific locus/gene
(more than one version of a gene)
how many polymorphisms exist in humans?
-abt 3 million differences per set of chromosomes! (6)
*how are polymorphisms classified? (what makes 1 allele unique from another)
single nucleotide polymorphisms (SNPs)
-a single base thats different
microsatellites (Msats)
-“satellites” highly repetitive; tandemly repeated
minisatellites (ministats)
deletions, duplications, insertions (indels)
-differences in size (PCR-gel electrophoresis)
single nucleotide polymorphisms (SNPs)
-single base pair substitutions between 2 homologous genes
(homologous: same ancestor; transition/ transversion)
most occur at anonymous loci (no known function;non-coding)
-does not phenotype (dont alter/ help)
most likely result of single mutation
-2 individuals with same SNP inherited from common ancestor
-2 diff mutations @exactly the same ____
used as DNA markers (landmarks along length of chromosomes)
-can trace to specific areas of chromosomes and use as landmarks to look for other genes
microsatellites
tandem repeats of 1 to 10 bases repeated up to abt 100 times
mututate bc of loss/ gain of whole repeat units via relication error (slippage)
-stutter
-change # of repeated units (add/subtract)
create polymorphism because of differences in length
microsatellites: replication slippage
replication “stuuters” when it copies repeats
new strand may loop put because of stutter: increases repeats= slip backward
template strand may loop out bc of sutter: repeats decreases=slip forward
minisatellites
same as microsatelittes but larger
20-100bp long, repeated up to thousands of times
create via replication slippage and unequal crossing over
(recombination- does not line up/ can add 1 homologue & delete 1 from the other)
Indels
deletion, duplicatiom, insertion of DNA in nonrepeating sequence
range in size from1 base to megabases
most are result of unequal crossing over and transpoable elements (TEs)
how do u detect differences?
what are some problems with this method?
isolate DNA, use PCR to amplify are where polymorphirm occurs, sequence, and compare between individuals
problems:
-expensive
-cant always ID heterozygotes
-time consuming for > a few individuals
how should the ID method be?
-inexpensive ( for large 3 of samples)
-fast (w/in a few hours-> days
-practical for large # of samples
what are the two ways to screen for SNPs?
if SNP alters RE site, can cut DNA and look for polymorphisms (create/destroy specific sequence)
need to know the SNP AND surrounding DNA
- southern blot
- PCR
SNPs- southern blot
-cut genomic DNA with RE affected by SNP
-run on gel & southern blot
-use of region of DNA near SNP as probe
*look at ppt
SNPs-PCR (best way)
-design primers complementary to sequence around SNP w affacted Re site (know DNA sequence that flanks the interested sequences)
-cut PCR product with RE
-run on gel and look for size differences
*this is how u ca genotype individuals for sickle cell anemia allele
(heterozygous: sickle cell syndrome)
SNPs: ASO hybridization
Allele-specific Oilognucleotide hybridization
use this method when SNP does not alter RE site
*listen again
ASO hybridization
* ppt
-PCR region with SNP
-blot PCR product on 2 membrane: replicates (split into half (+ & -)
-probe 1st membrane with short probe (20 bp) that matches 1 SNP allele
-probe 2nd membrane with short porobe that matches the other SNP allele
*only the probe w EXACT will bind to its respective SNP allele
probe 1 and 2 are different
sequence 1 &2 only different 1 single bp (know SNP)
*look over ppt
what is the quickest way detect DNA size differences? (microsatellites)
-PCR & gel eletrophoresis
(mutate by slippage-repeated tandemly)
-polymorphisms are differences in size
Minisatellites-PCR
whats its advantage over microsatellites?
adv: so large they can visuallize on agarose gel (>30 bp)
-possible to screen entire genome all at once
–>cut genome w REs run on gel, SOuthern blot, porbe with minisatellite sequence
-cant do this w micro bc of polyacrylamide gels
mini & micro satellite: banding pattern
* ppt
aka DNA fingerprint
=genotype pattern produced by simultaenous detectio of genotype at group of unlinked, highly polymorphic loci
-used in forensics and paternity analyses
Do gene location matter? when looking at polymorphisms
no, only size differences
what if you wanted to ID a specific gene associated with a specific trait, like disease?
if you know what protein is associated w trait/ disease, sequence it, infer DNA sequence based on aa sequence, screen lib
+hemophila A
-probe stick to it-> gene is present
finding a gene: hemophilia A
-researcher knew hemophiliacs had trouble clotting blod
-also knew proteins involved in clotting pathway
-knew they had 1 protein that was nonfunction in pathway: factor VIII (genetic cause)
-they isolated protein, sequenced it, inferred DNA sequence based on gentic coce, screen lib, isloated gene for factor VIII, dicvovered mutation that causes disease
what if you dont know what gene is affected?
-find gene by postional cloning
–> start by what chromosome carry the gene
+RECALL: if 2 alleles inherited more often than expected=linked
–>can use linkage of anonymous & disease-causing genes to located disase causing genes on a chromosome
(non-coding; usually no effect to phenotype
-MUST have a set of anonymous markers mapped to chromosomes (nned to know where anonymous markers are on chromosomes)
Mapping anonymous markers
*ppt
-make a mouse human hybrid
-mix mouse cells,human cels, sendai virus
-cell fuses tgt ( sendai virus- making many nuclei)
—> not all chromosomes are kept, some mouse and human chromosomes are lost
-after fusion, karyotype cells to determine what human chromosomes remain
-screen for specific anonmous makrers (usally PCR)
Q: did my anonmymous gene get amplified?
A: yes–> SNP is there (whatever chromosome has what ur looking for)
what happens after u map many anonymous loci?
-look at pedigrees of large families that have the disease
—>look for linkage between anonymous loci and disease
Q: do ppl w the disease also inherit a specific allele for the anonymous marker?
-once find linkage, sequence DNA between anonymous markers linked to the disease
-any protein coding between linked between markers is “candidate” gene
-once ID candidate gene, sequence them from diseased and non-diseases ppl
–if all diseased ppl have the same sequence at a candidate gene, and it differs from the sequence of non diseased ppl that is PRObably the gene associated with the disease
-to definitely test if this is the right gene, put it in an animal model
transgenic organisms
“across genome” from completely diff species
*genome is manipulated to carry foreign DNA
Q: what technique do u know of that creates transgenic bacteria?
A: molecular cloning and transformation
knock out organisms
-inject non-functional copy of gene in newly fertilized egg
-if mututated copy incorporated in host genome, passed om to offspring (become part of genome)
-researchers often make knock out mice to stoudy human diseases because mice and humans share many of the same genes
how to make transgenic organisms?
-test subjects are usually mice
-inject candidate gene (responsible gene) from the disease person in newly fertilized mouse egg (b4 gamete nuclei fuse)
—->if foreign DNA incorporated in host’s geome, it will be passes onto host’s offspring
*can use this technique to “knock out” function of gene that is normally found in genome of organism=knock out organism
transgenic organism vs knock out organism vs GMOs
transgenic: mutate a gene by distrupting the function of the gene that already existed in the genome (dna of one organism into another)
knock out: replace a functional gene that already exists of the genome into a nonfunctional one
