exam 3: distinguish individual genomes

Question 1

why would u want to distinguish individual genomes

Answer 1

*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

Question 2

ethical issues to who should have access to information and how should they use it?

Answer 2

-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

Question 3

polymorphisms

Answer 3

> 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)

Question 4

*how are polymorphisms classified? (what makes 1 allele unique from another)

Answer 4

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)

Question 5

single nucleotide polymorphisms (SNPs)

Answer 5

-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

Question 6

microsatellites

Answer 6

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

Question 7

microsatellites: replication slippage

Answer 7

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

Question 8

minisatellites

Answer 8

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)

Question 9

Indels

Answer 9

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)

Question 10

how do u detect differences?
what are some problems with this method?

Answer 10

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

Question 11

how should the ID method be?

Answer 11

-inexpensive ( for large 3 of samples)
-fast (w/in a few hours-> days
-practical for large # of samples

Question 12

what are the two ways to screen for SNPs?

Answer 12

if SNP alters RE site, can cut DNA and look for polymorphisms (create/destroy specific sequence)

need to know the SNP AND surrounding DNA

1. southern blot
2. PCR

Question 13

SNPs- southern blot

Answer 13

-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

Question 14

SNPs-PCR (best way)

Answer 14

-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)

Question 15

SNPs: ASO hybridization

Answer 15

Allele-specific Oilognucleotide hybridization

use this method when SNP does not alter RE site
*listen again

Question 16

ASO hybridization
* ppt

Answer 16

-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

Question 17

what is the quickest way detect DNA size differences? (microsatellites)

Answer 17

-PCR & gel eletrophoresis

(mutate by slippage-repeated tandemly)

-polymorphisms are differences in size

Question 18

Minisatellites-PCR
whats its advantage over microsatellites?

Answer 18

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

Question 19

mini & micro satellite: banding pattern
* ppt

Answer 19

aka DNA fingerprint
=genotype pattern produced by simultaenous detectio of genotype at group of unlinked, highly polymorphic loci
-used in forensics and paternity analyses

Question 20

Do gene location matter? when looking at polymorphisms

Answer 20

no, only size differences

Question 21

what if you wanted to ID a specific gene associated with a specific trait, like disease?

Answer 21

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

Question 22

finding a gene: hemophilia A

Answer 22

-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

Question 23

what if you dont know what gene is affected?

Answer 23

-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)

Question 24

Mapping anonymous markers
*ppt

Answer 24

-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)

Question 25

what happens after u map many anonymous loci?

Answer 25

-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

Question 26

transgenic organisms

Answer 26

“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

Question 27

knock out organisms

Answer 27

-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

Question 28

how to make transgenic organisms?

Answer 28

-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

Question 29

transgenic organism vs knock out organism vs GMOs

Answer 29

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