00:31okay so why don't we start it is nine
00:35go ahead and i'm sure some more people
00:37will trickle in as we
00:40go all right i need you to move a little
00:43so i can see the screen so on monday
00:48plan to have our i guess it's our third
00:51that's correct and we will
00:54have it over dna sequencing we change
00:57that light so it's not glaring
01:08that's a little better so we'll have it
01:13and um not about the history of the dna
01:16sequencing rivalry between solar
01:19genomics and the human
01:21uh genome project but about the
01:23sequencing chemistry itself the
01:28how the termination of synthesis
01:32leads to being able to actually read the
01:35how reading multiple sequences and then
01:37overlapping them allows you to produce
01:39the final sequence so that was the
01:42the concept of shotgun sequencing for
01:45um so more about the chemistry
01:48of the the process not the human aspect
01:51and then the second one second topic we
01:54haven't talked about today but it'll be
01:57that we i think check where we left off
02:01last time yeah that'll be the first
02:02thing we really talk about today
02:04which is the pcr reflex and how that can
02:09to genotype samples in this case we're
02:13thinking about genotyping the
02:14sabino horses but it can be used and has
02:16been used for a lot of different things
02:22we will get right into that here put the
02:27okay does anyone have any questions
02:29about what we've done
02:31thus far before we um before we continue
02:35with our paper uh i have a question dr
02:39yes um so at the last class meeting you
02:43using the example for cats and things
02:45about how like the cells spread through
02:48coat variations in them stuff what about
02:51calico cat or an animal that has like
02:53multiple colors more than like three or
02:58uh that's a good question so the
03:01the kit gene which is the one that's
03:04horses is responsible for the movement
03:09throughout the body so there's as your
03:13example right on cue so
03:16if you have an animal that um
03:21that has melanocytes throughout their
03:24all over their skin and uh that means
03:27they did not have a kit mutation
03:29that gives them the ability to produce
03:32multiple colors of pigment
03:37well let me show you an example
03:44i'm sorry reese just bear with me for a
03:47so reecey has the ability to make
03:51brown or black coat color
03:55uh pigment you melanin or fail melanin
03:58her back and her most of her face but
04:01she obviously has some
04:02mutation that has caused the melanocytes
04:05to not migrate all over her body because
04:06she has large patches
04:08of white fur too which if you notice are
04:11on the i guess ventral
04:15ventral surface and extremities just
04:17like in some animals that have a kit
04:20in some areas it doesn't matter that
04:23uh that she possesses the the version of
04:26the gene that makes brown or black fur
04:29she simply doesn't have melanocytes
04:30there so that when you see white on an
04:33there are no melanocytes in that region
04:36so it doesn't matter what other coat
04:38abilities they might have however if
04:41they have melanocytes throughout their
04:43then you have to ask well what other
04:45genes are now going to be
04:47involved in the coat color so now that
04:49they if you establish they have the
04:50ability to make pigment
04:52then we can ask well what version of
04:54mc1r do they have because that will
04:56red versus uh darker black or
04:59what version of the aguti protein which
05:01we haven't talked much about yet
05:03because that will determine whether um
05:06they make the dark form in certain areas
05:08and we can ask whether they have a gene
05:10that dilutes the pigment
05:12which is the next paper we're going to
05:13talk about as a dilution gene
05:15and also the cremello we talked about
05:17cremello the matte p gene causes
05:20so the calico 2 though is a little more
05:22of a complicated story
05:24and later in the semester we're going to
05:26look at x inactivation
05:28but you may remember from from genetics
05:31if you can remember that far back the
05:33olden days we actually
05:34met in class and humans talk to each
05:38but calico cats have that
05:41unique developmental issue where
05:45some at some point during the the um
05:49it's probably it probably predates the
05:51fetal period i think it would be
05:52still in embryogenesis one of their x
05:55chromosomes turns off in female
05:59and female cats one of the two x
06:01chromosomes turns off so let's say that
06:05turned off the x chromosome that they
06:08got from their father well let's say
06:09that their father was an orange cat and
06:12let's say their mother was a black cat
06:14well if they turn off the x chromosome
06:17from their father those cells are no
06:19longer going to be able to make
06:21the orange coat color because that was a
06:23gene they inherited on the x chromosome
06:26the cells that turn off the mother's x
06:28chromosome are not going to be able to
06:30make the black coat color because that's
06:31a gene they inherited from their mother
06:33and when x inactivation happens
06:35it's perpetuated from that point forward
06:37through development so all of the cells
06:44this always goes well
06:47grouchy cat there just grouchy because
06:51her legs are so pointy
06:54so if we fast forward to when this cat
06:56fully develops all of the cells
06:59let's say that these cells have the
07:00ability to make black and these cells
07:02have the ability to make orange
07:03wait i have the ability to make orange
07:10of course they're not going to be that
07:11color when they're in the embryo but
07:13eventually when they get to the stage
07:14where they're actually making pigment in
07:19the areas that are derived from cells
07:21that were expressing black
07:24will continue to express black so the
07:26cool thing about x-inactivation is once
07:28a cell makes a choice about which x
07:30chromosome is going to turn off
07:31of its two so this is a female specific
07:34process this doesn't happen
07:36in males once it makes that choice it
07:39decision for all of the subsequent cell
07:42cell once it's committed to turning off
07:45whichever it was the moms or the dads it
07:46always turns that off in all of the cell
07:49the daughter cells that follow so you
07:51end up getting patches
07:53in the animal that have either the moms
07:56x chromosome but not both and that's
07:59where you end up with your calico cat
08:02it is possible to have a male calico cat
08:05but it's extremely rare if you had a
08:08well like this is a female so she's xx
08:11you have to have two x chromosomes to
08:13a calico cat so if you were a male
08:17some of you probably remember uh what it
08:20well actually i'll just ask you what
08:22what's the syndrome where you have two x
08:23chromosomes and a y chromosome
08:27klinefelters blind filters so that's
08:31where in in cats this can also happen
08:33you can have this chromosomal
08:35abnormality in uh you know any organism
08:40sex determination system which is not
08:44but mammals at least
08:47so you can have a klein filter cat and
08:49they do you know occur every now and
08:51then i don't know what the frequency is
08:52i think it's something like 1 out of 10
08:55000 something extremely small
08:57but if it was a male calico it's likely
09:02abnormality which well for better or
09:05makes makes them infertile uh probably
09:08for better considering the cat
09:09population is out of control um
09:13but anyway that's a that's a long answer
09:16to your short question but it was a good
09:18and so the the thing to ask when you're
09:21talking about coat color
09:22for any organism is one do they have
09:25melanocytes in the area
09:28and if they do well if they don't then
09:31there's going to be white fur if they do
09:34have melanocytes then you can go down a
09:35different pathway and say well then what
09:39all these other genes that influence
09:44tyrosinase terp1 matte p agudi
09:49the them oh it's another one
09:53endothelium b that comes up in this
09:56so it's a matter of what if you can make
09:58pigment at all and then
10:00all the other genes that might then
10:02contribute to it so that's why you see
10:03so much variation in all these
10:04domesticated animals
10:06some people have bred for certain traits
10:10cats and dogs are one area where you see
10:14variation someone at a gas station
10:17recently tried to describe to me
10:21the genealogy of their dog i don't know
10:23why i don't think i seem like
10:25the type of person that someone would
10:26just walk up to and talk to about dogs
10:29i think he had a double doodle does that
10:33he tried to describe all these golden
10:35doodles he had that he had backbred to
10:37other poodles and things and somehow
10:39he ended up with what he called a double
10:41doodle i don't know what he was talking
10:44it was one of many strange moments in my
10:48okay any other questions who was it was
10:51alicia yes thank you
10:55you're welcome yes high five
10:59to hannah what's your question
11:02hi yeah um i i remember you said on
11:06wednesday that the piebaldism uh
11:08mutation does not cause
11:10um like detrimental effects in humans
11:13other than the differences in like
11:16um but like however i'm sorry was i
11:20um no i'm just saying i i was just
11:22saying like but in mice i remember you
11:24said it was different but it does
11:26cause like um detrimental effects like
11:29i don't remember what they were but you
11:31i think you listed off a few things
11:34um i think it was deafness
11:37maybe um yeah anemia
11:41sterility um like really sterile
11:44mast cell deficiency i think um
11:48but i was just wondering like why that's
11:49why that is the case like why
11:51it would cause a lot of bad things in um
11:54mice but not necessarily us
11:56i was wondering how they knew that the
11:58mice were deaf that's
12:00that's the question i had when i read
12:03uh i guess i guess you could figure it
12:05out but the mice usually don't talk so
12:07you can't really ask them questions
12:10i don't know why why they don't report
12:13issues in humans one explanation for why
12:17different in one animal versus another
12:19in general would be that
12:22that term we brought up a while back
12:24called a lilac heterogeneity where
12:26the mutation that causes all those
12:30might be a mutation in the gene which is
12:34than the mutation that occurs in human
12:38for the same gene so it could be a
12:39difference in that it could be that
12:42in humans if the gene is mutated
12:45that it's lethal and you never see so
12:48birth of the birth of children that have
12:51that mutation and only mild mutations
12:55are compatible with life so that's i'm
12:58speculating because i don't know the
13:00that's a possibility that it could also
13:04the piebaldism is caused by
13:08a homozygous condition so that they
13:10inherit two mutations because in mice of
13:13course these are all captive
13:14you know bred animals in a lab so they
13:16can they can maintain
13:18these inbred colonies and so if it was a
13:23issue that caused the the problem in
13:25mice it would be really unlikely
13:27in humans for two people with that
13:29mutation to have offspring just because
13:30of how rare it is so that's another
13:32kind of like well no that's not never
13:36mind i'm going to retract that example
13:37before i say it's not really a good one
13:40so those are some ideas i don't know why
13:42it would be so dramatic in one organism
13:44affect humans specifically but
13:48those those would be my my guesses
13:51my speculation okay thank you
13:55any other questions we've set up a
13:58a class record with two questions at the
14:00beginning this is awesome
14:06in retrospect i'm pretty pleased with my
14:14did pretty well for myself with that guy
14:19all right well if you have questions
14:23speak up while we're going along here
14:24but we might as well
14:26make a little forward progress oh
14:30a question last time about
14:33uh hold on a second about the exam and
14:37future quizzes and so forth so i did
14:40announce quiz monday we'll take care of
14:43third quiz on monday it'll just be open
14:44all day like the other quizzes and the
14:48so i'll work on that later and get it
14:51i think we'll just keep the exam on
14:53schedule so it was scheduled to be
14:58we'll have all of next week and then
15:00it'll be the following monday so that
15:02gives us today and three more lectures
15:04and i think that'll be fine to keep it
15:06on schedule that way we won't get too
15:08off track in our class we'll have plenty
15:10of material i mean the exam doesn't have
15:14you know an epic length so
15:17uh whatever we finished by next next
15:19friday or probably next
15:21wednesday and maybe just the very
15:23beginning of friday that'll be the exam
15:25material then we can spend the second
15:27friday or depending on where the papers
15:30most of friday doing some review and
15:33hopefully won't have any more
15:34weather incidents like last time so
15:37we'll just stay on schedule but
15:40syllabus quiz schedule that's simply to
15:44a reminder that we should be having
15:46quizzes here and there but i'll announce
15:49when they when they're actually going to
15:52okay let's look at our paper so we ended
15:55last time let me get all my windows
16:00in order here okay we ended last time
16:10on the beginning of paragraph
16:13or the the last sorry let me
16:17just start all that over we ended last
16:18time second column page 897
16:22with the paragraph talking about this
16:26a reverse transcriptase pcr now we're
16:29start talking about real-time pcr in the
16:31next paper and maybe we'll
16:33we might get into some of that today
16:34because we don't have too much left in
16:38but real-time pcr and rt-pcr are not the
16:41same thing they unfortunately share the
16:44acronym often when people talk about
16:47real-time pcr now to make that
16:49distinction they'll they'll say
16:50qpcr which is an abbreviation for
16:55but real-time pcr and quantitative pcr
16:58are synonymous rtpcr
17:02means reverse transcriptase pcr in this
17:06and it's worth noting since we're going
17:07to talk about quantitative pcr
17:10quantitative pcr is the
17:12definitive coveted test so if you take
17:15an antibody test which i don't think is
17:18as it used to be although it's it's
17:19rapid and fast so some people
17:21like it for that reason if you take the
17:24uh there's there are a decent percentage
17:28of false positives with that test which
17:30reason people shy away from it as being
17:35answer excuse me now but the the main
17:38nasal swab test that people take
17:40where you have to wait usually 24 hours
17:42before you see a result that is actually
17:46test so they're looking at the rna
17:52whatever tissue sample they get whether
17:54it's nasal or i think some people
17:56do saliva in some places um
18:00but any case they're isolating rna which
18:02is from the virus and then they're doing
18:04a quantitative pcr reaction to
18:06amplify that that viral uh message
18:10convert it to dna pcr amplify it and
18:12then tests to see its presence
18:14um so we'll actually talk about that
18:16technique it can be used for a lot of
18:18different things but that is what's
18:20if you get that long swab stuck up your
18:23that will be sent to a lab to do
18:28but this is not quantitative pcr so over
18:30here what we were doing
18:32or what they were doing was looking at
18:34reverse transcriptase pcr taking
18:36messenger rna from these horses and
18:39converting the messenger rna to dna
18:42doing a pcr reaction with that dna to
18:45of the bands and specifically they were
18:48doing a pcr with primers that annealed
18:52to exon 16 and exon 18
18:58well which is then converted to dna
19:01so that means if you have a 16 17 18
19:05span you'll have a large product which
19:09here's my little pointer which is what
19:13here with the 356 base spare piece if
19:17the absence of exon 17 which is the
19:20theme of the paper right exon 17
19:22skipping in the horse
19:24if you had the absence of exon 17 then
19:26you're going to have this smaller
19:28and the results that they found was that
19:33heterozygous for the mutation
19:36had two bands horses who were homozygous
19:39also had two bands which meant they were
19:42skipping in some cases
19:43but what they found is that the ratio of
19:45bands was different depending on whether
19:47you were homozygous or heterozygous
19:49so the more mutation copies you had
19:52meaning homozygous the more likely you
19:55skip exon 17 and we'll talk about why
19:59in a second if you're heterozygous
20:01you're likely to have both transcripts
20:03the majority of yours of the horses
20:07were the functional transcript including
20:09exon 17. if you were homozygous for the
20:11mutation the majority of the transcripts
20:14were absent of exon 17
20:17which of course is a problem okay so
20:19that's where we ended
20:21let's keep chugging along here to figure
20:24out what was going on
20:26now that they have seen this phenotype
20:31recognized that it's a splicing issue
20:33potentially they want to know
20:34what actually is the issue why why are
20:37they skipping exon 17 in some of these
20:39animals and not others
20:43so to do that they start looking at the
20:45dna to see if they can find a mutation
20:48can be associated with this
20:51phenotype now fortunately in this case
20:53it's a lot easier than what we saw in
20:55the first two papers they don't have to
20:56go searching for the gene
20:57they've already got the gene in hand
20:59they know it's something to do with kit
21:02and so that they don't have to do a
21:05so knowing that it's something in the
21:08exxon 17 that's going on they open up
21:13investigation into this by looking at
21:17exon before and the intron and a little
21:21bit of the exon afterwards i believe
21:24and so they look at 290
21:27bases of the estimated 5 000 total in
21:31intron 17 were amplified in sequence for
21:34uh sabino's and eight non-sabino horses
21:37within the excuse me 1049 bases
21:42seven snips were found but only one
21:46was present in all four of the horses
21:49producing these aberrant transcripts so
21:52they do find seven variations
21:54but when they do an association
21:56comparison between which has the
21:59and which has the particular snip they
22:01narrow it down to one
22:03okay and so the site that they find
22:06we've already looked at this
22:07naming scheme which is kind of
22:09complicated but uh was designated ki-16
22:131037 which was the kit gene
22:16intron 16 position plus 1037 meaning
22:22from the start of transcription for that
22:26the transcriptional start site is always
22:29so 1037 bases later they have this
22:33snip and it's in an intron
22:36and the mutation is a t to a
22:39so that's a transversion mutation
22:42because you're going from a
22:43pyrimidine to a purine so it's a t to a
22:47and notice the location of this 13 base
22:50upstream of exon 17.
22:54so uh this is still on the intron but
22:58just slightly over a dozen nucleotides
23:01upstream of where the next exon begins
23:05okay so it's right in the vicinity it's
23:08not at the splice site
23:09but it's very close to that supply site
23:13now this the fact that it resulted as
23:15they say in the elimination of an mnl1
23:17restriction site this has no biological
23:21the horse okay the horse dna just like
23:25everything's dna has restriction enzyme
23:29right restriction enzyme sites are
23:30simply short sequences that a particular
23:33enzyme can recognize and cut so it
23:36doesn't since horses don't make
23:37restriction enzymes themselves
23:40change in a restriction enzyme site
23:41doesn't affect its biology
23:43the reason this is significant and i
23:45mentioned that is it provides a tool for
23:48to distinguish between dna that does
23:51and doesn't have this mutation because
23:54if the dna does have the mutation
23:56i'm sorry let me back up if the dna does
23:58not have the t to a substitution
24:01the enzyme mnl1 will cut that dna
24:05if it does have the substitution that
24:08that is recognized by the enzyme is
24:11so you can distinguish between horses
24:13that do and don't have the mutation
24:15some of them you can cut their dna
24:17readily and some of them
24:19you cannot okay so they say this allowed
24:21for the use of a pcr rift flip
24:25to quickly screen individuals for the a
24:28and they also mentioned just because
24:31people probably will
24:32people reviewing this paper before
24:34publication might have recognized there
24:35were other snips in that region
24:37so they do just complete their story
24:39they say other stumps were identified
24:41but they didn't show association with
24:43the skippy so how does this pcr rifflip
24:53methods let's find where where are the
25:01horses horses horses
25:08i must have skipped over
25:13there it is okay so if we look at this
25:20well let me read it first and then i'll
25:24i'll discuss it a little so they say
25:28to screen for the polymorphism
25:31the portion of three prime end of exon
25:34intron 16 in the beginning of exon
25:3617 was amplified using pcr and they give
25:40specific pcr reagents they use show the
25:44a tda substitution eliminates the
25:48um mnl one site when the sabino allele
25:52and so they've designated someone asked
25:54me this in a an email question which
25:56mutation was which so the the wild type
26:00sb1 remember the wild type is recessive
26:04the mutant allele even though the allele
26:07is not completely dominant when it's
26:10present as a heterozygous case
26:12it's semi-dominant or incompletely
26:16but that doesn't mean that sb1 wild type
26:20is semi-recessive so the sb1 is still
26:23recessive regardless of whether the
26:25other allele is fully dominant or
26:27incompletely dominant but the mutation
26:29is uppercase because it is
26:32incompletely dominant it is it's still
26:35in the dominant category so we call it
26:37sb1 with capital letters
26:40the pcr product also is was designed to
26:43include an additional ml
26:44in l1 site to be used as a positive
26:49okay so let's talk about what this
26:58experiment actually does so if you
27:01well i don't know if you're scrolling or
27:02if you have a paper copy or if you're
27:04watching mine but if you are following
27:07along in a paper copy
27:13page 898 so i'm going to unshare my
27:17because i want to draw this out instead
27:22just reading it one second
27:33all right so the pcr rifflip is a
27:36great assay because it's very easy to
27:40and the riflip acronym is
28:01restriction fragment length polymorphism
28:04this was one of the earliest
28:05assays to uh to arrive at what we
28:09sometimes called dna fingerprinting and
28:13the 80s someone developed a method to do
28:18where before pcr became widespread or
28:21was he actually probably before pcr was
28:24um by carrie mullis i think that was
28:28mid to late 80s but anyway what what
28:31scientists did was they took genomic dna
28:34and they cut it with a restriction
28:35enzyme and it would produce lots of
28:38right as any dna would but what they
28:40found was that because of
28:42differences from one human to the next
28:45the same enzyme would produce a
28:46different set of fragments
28:48say in me that it might produce in in
28:50one of you because we're not related
28:52and so just little snips throughout the
28:55if they happen to occur in restriction
29:00would determine whether one person's dna
29:02was cut at a certain place
29:03or not so if you compare the person's
29:10you know you might get a set of bands
29:12like this for one person you might get
29:15a separate set of bands for another
29:17person and let's say that this was
29:19uh the mother and the father you then
29:23whatever their offspring
29:27if they had offspring the offspring
29:29would have a combination of bands
29:31from each of the parents right they
29:33wouldn't have all of moms and olive dads
29:35but they would have 50 of their you know
29:38polymorphic sites would be from one
29:40from the other give or take a little
29:43depending on crossing over and all kinds
29:45subtleties so this was one of the early
29:48ways that people could test
29:49for um for familial relationships and
29:53the guy that developed this technology
29:56uh originally was in um
29:59he was in uh not london he was in
30:02england it wasn't london
30:04i can't think of the name of the city
30:08anyway he developed this technology
30:11he was trying to help um british
30:15who were returning from outside of the
30:16country to establish that they were
30:19you know born in britain by comparing
30:23relatives so it was sort of a paternity
30:25test only it wasn't a case of
30:28unknown paternity it was just to
30:29establish you are a blood relative
30:32or you were born here or whatever but
30:35what took place later and some of you
30:37some of you have had
30:39the capstone class and we talked we did
30:41a talk about this in capstone
30:44um later it became the first
30:47technology to actually
30:51find a murderer using dna evidence so
30:55dna samples taken from
30:58two rape victims and they actually
31:00convicted the murderer whose name was of
31:02all things colin pitchfork
31:05but that was a an example of restriction
31:08fragment length polymorphisms
31:10designating one individual uh from
31:13now when you do pcr riff flips it's a
31:18and so a pcr reflex let's say that we
31:26of a gene and so this is one let's say
31:29heterozygous so here's the plus
31:32the wild-type chromosome and here's the
31:34mutant chromosome we could do pcr
31:39of the region where this mutation occurs
31:44we could produce you know lots of copies
31:51of these uh these regions some would
31:53have the mutation and some would not
31:55well if that mutation happened to occur
31:57at a place where restriction site was
32:00then if we digested this dna using a
32:05a restriction endonuclease if the wild
32:08dna has the enzyme site that dna
32:14will be cut into pieces if the mutation
32:18removes that restriction enzyme side if
32:19it changes the sequence so the
32:21restriction enzyme no longer cuts
32:26that dna will remain unaltered so you'll
32:28get a different result depending on
32:29whether you have the
32:31the mutation or whether you don't so
32:34uh the polymorphism the different result
32:37from the restriction fragment
32:38the difference in the original you know
32:421980s version and this is that now
32:44you've introduced pcr
32:46and the the reason why that's
32:47advantageous is we don't
32:49want to produce you know 10
32:52000 fragments from their entire genomic
32:55sample and then find the single fragment
32:58that might be different that would be
33:00instead we just want to look
33:02specifically at one section
33:04one section alone and find out if a
33:06single nucleotide change has occurred in
33:08that section so on the paper
33:10on page 898 they describe precisely
33:14what they expect to get
33:19and so let me draw their case i think if
33:22we look at the specifics of their case
33:25make more sense so i made a little chart
33:28for myself with the numbers
33:30there is one number that doesn't quite
33:31add up which i'm not sure
33:33if it's just a typographical error or
33:37so in the wild type allele which
33:44they say in the paper that the um
33:50the sb produced bands 207 74 and 47 base
33:56whereas the sabino white individuals
33:58produced bands 252 and 74
34:01in size so this is our pcr
34:04product this is the region that they're
34:05talking about we have
34:09the mutation and we have the wild type
34:13both of them initially produce the same
34:17size band just from pcr so this is our
34:19region and it spans from
34:22um it's at the end of
34:25the intron in the beginning of the exon
34:27so the pieces that they discuss
34:29if we write them out one was 74
34:33one was 207 and one was
34:3747. so that means there is an enzyme
34:41site that cut at those actually i'm not
34:43going to make an x because x is usually
34:45implied in mutation let's just make a
34:50so there were two mnl1 sites in the wild
34:54version of this sequence okay but
34:57when they look at the sabino mutation
35:00they say that the products are two bands
35:04and 74. so in the sabino
35:08you still have this 74 base pair but
35:12you have something that's 252.
35:16now if you're looking at the numbers
35:17well first of all what's happened
35:20is that the mutation for the t
35:23to a substitution occurs at a place that
35:25eliminates that second site
35:27so the sequence for the mnl1
35:32recognition is kind of bizarre if you
35:33looked at the paper but
35:35anyway that one nucleotide changes that
35:39so it can no longer be cut so now this
35:42sequence which initially had been cut
35:44into two pieces is now
35:45only going to end up being one piece
35:47okay the enzyme doesn't cut it anymore
35:50if you're doing the math though you
35:51might notice these two things should add
35:53up to 254 so i'm not sure where the two
35:55nucleotides went i've always wondered
35:57that i think it might just be
35:59a typographical error but in any case
36:04so what they can do with their horses
36:07they can take dna from the horse and if
36:12was a wild type horse so little
36:17sb1 slash little sb1
36:22i'm gonna need more room cat i'm sorry
36:35okay so here's what we would expect to
36:36see we can have our wild type horse
36:38we can have our heterozygote
36:45and we can have our homozygous
36:52so these three possibilities if we have
36:55we expect to see three bands okay so the
36:59a small band at 47 base pairs
37:04so you use pcr with dna from that horse
37:08you produce a fragment
37:11you digest the fragment mln1 and you get
37:15the 47 base pair piece a 74 base pair
37:20and a 207 base pair piece
37:25all right now in a horse that is
37:28they should produce a pcr product for
37:30both of these alleles
37:32and then when you cut them they should
37:34produce the fragments that we
37:36see in all of these so we'll still see
37:38everything we saw in the original
37:41lane here but now we should also see an
37:44additional band because if the mutant is
37:46we have the 74 base pair so
37:50the 74 from both of these alleles is
37:52going to show up at the same spot right
37:55you know we'll just see one banded
37:57that's from both horses
37:58but this band wasn't present in our
38:00first lane 252 so that's going to be
38:08now what about the homozygous mutant
38:11this one produces a 74 base pair band
38:15and a 252 base pair band but it doesn't
38:20so with this simple assay and again this
38:23when i say excuse me when i say
38:26simple i mean that quite literally it's
38:29a it is a simple assay this is
38:31just a morning's work you can isolate
38:35from a whole from a blood sample in less
38:38than an hour you can do a pcr in about
38:41restriction digest now takes about 20
38:44and you can run a gel in about 30
38:46minutes so you could if you were
38:47efficient you could be done with this
38:49assay by lunchtime so really
38:53much easier than sequencing for example
38:55or some of the other assays
38:56people use and very inexpensive
39:00very inexpensive to do this um but you
39:04difference between these so when they
39:05did this in their horse sample let's go
39:11when they did this in their horse sample
39:20there it is okay same page all right
39:23when they do this in the horse sample
39:25that they are using make a little easier
39:28to read so the first lane
39:38this will go really well
39:46all right and the homozygous mutant if
39:47we look back on that's probably too
39:49small for you to see
39:51maybe not the homozygous mutant should
40:03and there is 74. we'll deal with this
40:07stuff at the bottom here in just a
40:09all right the second lane is
40:12um i'm going to write pluses and minuses
40:15because that'll make it much easier so
40:17homozygous mutant is minus minus the
40:24so they have one copy of the little s
40:27one allele and one copy of the big s big
40:30allele so they're homozygous for the
40:32mutation so they should have and they do
40:35a band at 252 and a band at 74.
40:39but they should additionally have
40:42diagram a banned at 207 and a band at
40:46so there's a band at 207 and there's a
40:50i know you see something in lane two
40:52here and you're saying but
40:54it's there also just be patient i'll
40:57explain that all the way in a second
40:59all right and then the fourth case or
41:01lane four rather not the fourth case
41:03this is a homozygous wild type so little
41:081 and little sp1 and they should have a
41:13207 74 and 47. so they
41:16lose the band of 252. now you may see
41:20actually a very faint band here
41:23or at least i see a very faint band here
41:27just when they load sometimes when you
41:29load a lane a little bit will spill
41:32into the neighboring lane so sometimes
41:33you'll see a little ghost band where a
41:36has just diffused into there but that's
41:38definitely not a positive result
41:41okay and then uh what was lane five
41:44oh lane five was where they did the pcr
41:49but they did not use um
41:52the restriction enzyme so this is uncut
41:54dna just to show what the full band
41:56would be so the full band
41:57would be 326 right right
42:01yeah 326 so that's just a positive
42:04control that the pcr worked and then
42:07is a negative control
42:10where they don't do the pcr reaction
42:14so most likely what they've done let's
42:16see if they say it in the
42:20they don't say exactly how they did the
42:22negative control but they probably just
42:24the tac polymerase so they put in all
42:26the reagents except the polymerase
42:28and that's that so it's important that
42:31we see these two last lanes because it
42:34what's going on in this bottom lane so
42:38should be present whenever the wild type
42:42dna is present so it is
42:44the question is why is that thing
42:45present in the other three lanes where
42:47we clearly clearly see it
42:49and the answer is that it is not that is
42:51not the 47 base pair
42:53pcr rifle fragment we're looking at in
43:03are these are all unused
43:13it must be such torture to watch me
43:17with the mouse when i do these are
43:22okay and the reason that we have unused
43:24primers you always add
43:26primer in excess of what you need so
43:28that that doesn't become a limiting
43:30reagent in your pcr reaction the primers
43:33are about 25 nucleotides long in fact i
43:36checked in the methods i think there
43:40nucleotides or 25 nucleotides right
43:43so those are just close enough to the
43:46length of 47 that on a gel
43:48of this size they don't separate enough
43:50to see the difference
43:52if we ran this gel if we had a much
43:54larger gel and we ran it for a much
43:56longer time you'd eventually see those
43:59start to separate and be um be resolved
44:03um but this is just a small compact gel
44:06and they haven't run it very long
44:08so that's what it is you're seeing the
44:10overlap of the 25 nucleotide primers
44:13that haven't been used in the reaction
44:17with the 47 base pair piece so that
44:20bottom lane just is our
44:21line just isn't very it's just not very
44:25we could get all the information we need
44:28just by looking up here
44:29and another uh reason to assume that
44:34that these are the primers is how bright
44:36it is in this last lane where the pcr
44:37reaction didn't occur at our all
44:39none of the primer was used up and
44:42converted into the product
44:44i remember in pcr unlike dna replication
44:46in the cell the primer doesn't get
44:49degraded after the fact the primer just
44:51becomes part of the product
44:53you add on to the end of the primer and
44:55then the primer stays doesn't go
44:57so the more successful the pcr is for
45:00example look how bright this band is in
45:03and look how faint the primers are so
45:05most of those primers
45:07became part of the product where there
45:11the primers are much brighter and the
45:13brightness that occurs in a gel
45:16um i don't i don't know if this would be
45:19obvious or intuitive or not but when we
45:22see a bright band in a gel what we're
45:24is dna that has absorbed a fluorescent
45:28uh stain or dye and so the brighter the
45:32band that we see the more diet has
45:35uh the fainter the band the less diet is
45:38absorbed and the absorption of
45:39dye is proportional to to the size of
45:43so if we have this is getting
45:47so sloppy now i need to maybe i'll use a
45:55these two things for example they're the
45:56same size but quite a bit more
46:02than here and one explanation for that
46:05in lane four this is a homozygous horse
46:09so the amount of template material is
46:11twice as much because they have two
46:13copies of the allele
46:14and you probably have hundreds or
46:17of of you know each you probably have
46:20hundreds of thousands of
46:22cells worth of dna each with two copies
46:25in lane three you have half as much dna
46:29because it's heterozygous for that
46:32allele but any case the point is if you
46:35have half as much dna it can absorb half
46:38and so it's going to be half as bright
46:40so that brightness does
46:41sort of uh reveal the
46:45the concentration of dna that we're
46:46looking at now it's not perfectly linear
46:49uh there are lots of other factors but
46:51it's close it's a good indicator
46:58what pcr rifflip is and how they did it
47:01and what did they find
47:09okay so they they said that they um
47:12it correlated there so they did an
47:14association study now to see does this
47:17with a larger family so they looked at
47:19uh some walking horses
47:21all three families sabina was only
47:23spotting pattern present
47:25there are some other um white patterns
47:31and we know the the cream or cremello
47:35were not observed and their associated
47:39rifle alleles were not detected so they
47:41also made a pcr riflip
47:43test for those other colors so these
47:47i had to look these up because i wasn't
47:50oh dear what have i done
47:56so if we look at the frame of arrow
48:00it's a pretty horse there and if we look
48:04so let's compare that to the sabino
48:07pair of sabino's and if we compare that
48:15i have a picture of the
48:21oh no that is the one i missed okay so
48:24so they're pretty similar they all are
48:25patchy white um and so they did a pcr
48:29riff flip test for each of those because
48:32mutations and they found that
48:36um that they were not observed in their
48:41so then they expand this is kind of like
48:43the tiger paper they expand
48:45their analysis further the total of 320
48:48horses from 13 breeds
48:49were tested for this snip now again this
48:53320 horses sounds like a lot of dna to
48:55deal with but it's really not you can do
48:59this would still be just a couple days
49:01work if you were organized
49:04i guess the hardest part is just getting
49:05the blood in the first place
49:08but horses are pretty of course they
49:13dr prater drew some horse blood for me
49:16as a as a good friend would uh years ago
49:21remarkable to see how accurately he
49:23could find that vein in their neck
49:25um but i guess you've done it for
49:27decades you get good at it
49:29anyway total 320 horse horses were
49:32tested and what did they find
49:34the snip was present among a handful of
49:39and all breeds horses homozygous for the
49:42were white among horses that had a
49:46uh allele all were either sabino
49:50or white and horses that were
49:53phenotypically white
49:54so here's an important thing that at
49:56first would look like a
49:59a glitch in your data horses that were
50:01phenotypically white but heterozygous
50:04also tested positive for other patterns
50:08so if a horse looked like a homozygous
50:12but only tested as a heterozygote then
50:15they found that this horse
50:16had a separate mutation that was causing
50:20it to be completely white so the tobiano
50:25the olws is the ovaro pattern it stands
50:32white foal syndrome because in its
50:35homozygous state this mutation
50:37um the horses are born uh
50:40but they die within a few days because
50:44that particular mutant causes a um
50:47a dysfunction in the development of
50:49their digestive system so they can't
50:52and uh they can't digest it they can't
50:56they just the food builds up inside them
50:59they uh eventually die really there's
51:02and there's not nothing you can do
51:04to fix it at least from what i read
51:07so it appears that there's an additive
51:09effect between sp1 and other white
51:12this is important it's a good example of
51:15genetic heterogeneity
51:17where more than one gene can lead to the
51:20or similar phenotype so in this case
51:24multiple different avenues to arrive at
51:27the white spotting or the total white
51:31in these horses okay so let's see what
51:39i'm not sure the rest of that was
51:41significant they did look
51:44at other horses that had the similar
51:46phenotypes and found that in some cases
51:49they did not see the same exon 17
51:53again they're talking about uh just the
51:56genetic heterogeneity
51:57now that they already mentioned okay so
52:01matter to the gene in the first place
52:02this is a single nucleotide polymorphism
52:05it's an intron it doesn't even make it
52:08into or shouldn't even make it into the
52:11uh region of the final process mrna so
52:15well they look at this that mutation and
52:18they analyze it compared to other
52:20intronic sites to ask why why is it
52:25and so they find that the
52:28i think this is kind of funny so when
52:32to 2005 they call it an online applet
52:36no one says applet anymore which means
52:39little application just say app but
52:43it sort of dates the paper uh but the
52:46splice score for both the mutant and the
52:47wild type sequence was calculated
52:50using an app based on a formula
52:52developed by a couple folks
52:53in a previous publication what they're
52:56doing when they say they're
52:57the splice score they're asking compared
53:00ideal splice site consensus sequence
53:04how far off is this mutation
53:07how would this affect what would be
53:09considered the ideal splice sequence
53:11at the end of an intron and so they do
53:14they have some numbers here and again
53:15these numbers don't mean anything
53:17unless we had a comparison with other
53:21application but uh the importance i
53:25the relative comparison and so the wild
53:28type site was stronger than the a
53:30containing site with scores of 79
53:32and 77 respectively and so
53:36again unless we had a number of of
53:38different things to compare it to using
53:40the same app we can't really
53:42say much about that but they do go on to
53:47i thought was more important let's see
53:48if i marked in my paperwhere
53:53here we go this this paragraph is a
53:55little better to discuss that final
53:57data blip that they made okay so we know
54:02this mutation is 13 base pairs up from
54:06a typical splice consensus sequence is
54:08relatively small right
54:10it's about six or seven uh base pairs
54:13i think we oops sorry
54:17i think we talked about it i don't know
54:18if you can still see my screen i'm
54:20trying not to flip back and forth too
54:22but the typical splice consensus site is
54:30exon intron junction
54:33so this would be the exon and this would
54:38but it's that cagu and it cuts between
54:42that is what's uh often considered to be
54:44the splice consensus
54:46so the mutation we're talking about is
54:49just over a dozen nucleotides upstream
54:52of where that splice consensus so it
54:54seems like it wouldn't have any effect
54:56but it does clearly have an effect and
54:59they look at other genes and other
55:05the part i think is useful evidence for
55:07this notion is supported by the
55:10at this position for other genes and
55:11species in a study of introns among
55:14the position 13 bases before splice
55:18t 41 of the time while a was the least
55:21common occurring only five percent of
55:24keep in mind there are only four options
55:26for a nucleotide right
55:27c a g or u in the rna at least
55:32and so if a t is present 41 of the time
55:35that you know 25 would be average
55:39so 41 of the time it's almost twice as
55:42as it would be under just average
55:45uh oh jeez why am i still talking it's
55:50why didn't anyone tell me that uh that
55:53we're way over time okay we'll pick this
55:55last time my apologies for going over i
55:59hate when teachers do that and i
56:01sorry that i did all right we'll pick it
56:02up next time you have all the
56:04information you need for the quiz
56:06we will finish this paper in the first
56:08few minutes of our next meeting and i
56:10a great rest of your day my apologies