Hello. Attached I have 3 genetic labs that I need assistance with. In some of the questions, it says you'll need to watch the video to answer the questions. This for the most part is not true. I usual

N ATIO NAL C EN TER F O R C ASE S TU DY T EA CHIN G IN S C IE N CE

P art I – F lu ore sce nt d dN TP s

Sara , a co lle ge so phom ore m ajo rin g in b io lo gica l sci ence , w as both e xci te d a nd w orrie d. S he h ad b een a w ard ed a

r e se arch in te rn sh ip in a la b w hose p ro je ct w as to fin d th e sp eci es co mposi tio n o f a st udy site n ear ca mpus. H er m ain

a ct ivi ty would b e th e D NA se quenci ng o f sp eci fic genes use d fo r sp eci es id entifica tio n. B ut it h ad b een a w hole ye ar

si nce sh e fir st le arn ed th e to pic of D NA se quenci ng fr o m h er in tr o duct ory bio lo gy class; h ow m uch d id sh e r e ally

r e m em ber? S o, o n a T hursd ay afte rn oon, sh e o pened h er o ld n ote s and r e vi ew ed th e p ara gra phs sh e h ad w ritte n a bout

S anger se quenci ng:

T he m ost w id ely use d D NA se quenci ng te ch niq ue w as in ve nte d b y Fre derick Sanger b ase d in C am brid ge, E ngla nd,

and h is co lle agues, P aul B erg a nd W alte r G ilb ert. T hey use d th e syn th esi ze d d id eoxyn ucl eotid es to te rm in ate th e ch ain

o f D NA e lo ngatio n. T his new te ch niq ue is base d o n th e se le ct ive in co rp ora tio n o f ch ain -te rm in atin g d id eoxyn ucl eotid es,

also kn ow n a s 2', 3 ' d id eoxyn ucl eotid es, a nd a bbre vi ate d a s ddN TP s

(d dG TP, d dA TP, d dT TP a nd d dC TP ). T he e xt ensi on r e act io n is divi ded b etw een fo ur d iffe re nt te rm in atio n r e act io ns,

each co nta in in g a r a dio act ive n ucl eotid e th at ca n b e in co rp ora te d b y th e D NA p olym era se d urin g th e e xt ensi on st ep.

B eca use th e d id eoxyn ucl eotid es la ck a h yd ro xyl g ro up o n th e 3 ́ ca rb on o f th e su gar r in g, th e a dditio n o f a n ucl eotid e

ca nnot co ntin ue, a nd th is m arks th e e nd o f th e e lo ngatio n. T he r e su lt- in g fr a gm ents ca n th en b e r e so lve d o n a h ig h-

re so lu tio n fo ur-la ne-w id e a nd r a dio act ive p olya crylam id e g el th at r e ve als th e co mple m enta ry se quence ( F ig ure 1 ).

S anger, B erg a nd G ilb ert w on th e N obel P rize o f C hem ist ry in 1 980 fo r th e “ d id eoxy meth od” a lso kn ow n a s “S anger

m eth od” o r th e “ fir st g enera tio n se quenci ng” ( se e S anger e t a l., 1 977).

L ate r, P ro fe sso r H ood’s te am a t C alte ch d eve lo ped th e te ch niq ue, in w hich th e r a dio act ive n ucl eotid e w as re pla ce d b y

flu ore sce nt la bels and in cl uded th e u se o f a la se r to d ete ct th e flu ore sce nt co lo rs. T he e le ct ro phore tic pro ce ss is

co nnect ed to a co mpute r fo r th e d isp la y of th e r e su ltin g se quence . T hey publish ed th eir w ork in N atu re ( se e S m ith e t a l.,

1 986).

T his work was fu rth er im pro ve d b y th e flu ore sce nt la belin g o f d dN TP s by a te am o f r e se arch ers at th e D uP ont R ese arch

S ta tio n a nd th e te ch niq ue w as descr ib ed in a n a rticl e le d b y Dr. Ja mes Pro ber in th e jo urn al S ci ence ( se e P ro ber e t a l.,

1 987).

S ie rra N etz -D ec 03, 2 021, 1 1 :0 0 A M M ST Figur e 1 . A utor adi ogr aph f rom four sepa ra te di deoxy s eque ncing r eactions .

On h er fir st d ay of r e se arch w ork, S ara m et w ith th e p ost doct ora l r e se arch er, D r. D ana, w ho w ould g o o ve r th e d eta ils of

her ta sks with h er. D r. D ana sm ile d a t h er n ew in te rn . “ W elco me S ara ! A re yo u e xci te d fo r yo ur fir st d ay in la b?”

“H i, D r. D ana,” S ara r e plie d. “ I’m r e ally exci te d to g et st arte d, a lth ough I’m a little w orrie d b eca use I’ve n eve r d one

se quenci ng e xp erim ents befo re . A lso , I’ve n eve r u se d th ose se quenci ng m ach in es, so I’m a b it a nxi ous. ”

Dr. D ana n odded w ith u nderst andin g. “ W ell, I h ave g ood a nd b ad n ew s. I’ll st art w ith th e b ad; o ur se quenci ng m ach in e is

u nder r o utin e m ain te nance to day, b ut it sh ould b e w orki ng b y to m orro w m orn in g. T he g ood n ew s is th at yo u w ill n ot

w ast e yo ur tim e to day, b eca use yo u’ll b e p re parin g th e r e act io n tu bes to b e u se d to m orro w .”

S ara r e ce ive d th e p ro to co l a nd th e m ate ria l fr o m D r. D ana a nd st arte d h er b ench w ork. S he w ro te in h er la b n ote book

th e in gre die nts fo r th e e xp erim ent. T he D NA sh e w as tr yi ng to se quence w as 3 ́A TC GGTA C AAG GTC G5 ́ w ith th e fir st

five b ase s use d fo r p rim er b in din g. Exp erim ent 1 :

• 0.5 pmole DNA template

• 0.5 pmole primer

• 1 mL of 10× Taq polymerase

• 80 mM dNTPs (equal portion of dATP, dTTP, dGTP, dCTP)

• 8 mM ddNTPs each

-

-

-

-

Total: 10 ml volume

A s sh e g ently added a nd m ixe d a ll o f th e co mponents to geth er S ara w ondere d w hat w as act ually happenin g w ith in th e

tu be. S he w ent o nlin e a nd w atch ed th e fo llo w in g vi deo to r e m in d h erse lf o f th e p rin ci ple s of D NA se quenci ng:

S ara ca lle d D r. D ana to le t h er kn ow th at sh e h ad fin ish ed p re parin g th e tu be fo r cycl e se quenci ng. D r. D ana w as clearly

p le ase d. “ T hat’s gre at, S ara , yo u’r e fa st ! W e’ll p ut th at o n th e th erm al cycl er a nd in th e m eantim e r u n a tr a ditio nal

se quenci ng, ju st to g et a n id ea o f th is sh ort se quence . W e’ll th en co mpare th is to th e ch ro m ato gra m to m orro w o nce th e

m ach in e is up a nd r u nnin g to co nfir m th e se quence . W hat d o yo u th in k? ”

“S ure ,” r e plie d S ara . “ B ut fir st , w hat d o yo u m ean e xa ctly by ‘tr a ditio nal se quenci ng’? ”

“O h! I’m su re yo u’ve st udie d th at b efo re . S equenci ng g els have n’t a lw ays been co lo rfu l. In th e o rig in al S anger

se quenci ng m eth od, n o o ne co uld te ll o ne fr a gm ent a part fr o m a noth er. S o, in o rd er to b e a ble to fig ure o ut th e

se quence , p eople h ad to m ake su re th e fo ur r e act io ns were se t u p in fo ur se para te tu bes and th ey had to kn ow w hich

tu be h ad w hich d id eoxyn ucl eotid e.”

DN A Sequencing: The Chain T ermination Method (Sanger Method)DNA Sequencing: The Chain T ermination Method (Sanger Method) Sara n odded. “ S o, d o yo u m ean I’ll h ave to p re pare fo ur d iffe re nt tu bes and lo ad th em in fo ur d iffe re nt w ells la te r? ”

“E xa ctly ,” r e plie d D r. D ana. “ A nd p le ase m ake su re yo u w rite e ve ryt hin g yo u d o in yo ur n ote book. ”

Sara h eaded b ack to th e b ench . S he u se d th e sa me te m pla te D NA 3 ́A TC GGTA C AAG GTC G5 ́ w ith th e fir st five b ase s

use d fo r p rim er b in din g. S he st arte d h er w ork but w as a little d ist ra ct ed b y th e m usi c sh e w as list enin g to . W hen sh e

notice d th at sh e h ad m ade a m ist ake in th e p ro ce dure , sh e r e co rd ed th e e rro r in h er n ote s:

E xp erim ent 2 :

• 0.5 pmole DNA template

• 0.5 pmole radioactive primer

• 10 mL of 10X Taq polymerase

• 80 mM dNTPs (equal portion of dATP, dTTP, dGTP, dCTP)

• 8 mM ddNTPs each, separately

- ddATP** (Oops! I forgot to add ddATP in the A tube!)

- ddGTP

- ddTTP

- ddCTP

Total in 10 mL volume

A nsw er t h e q uestio ns b elo w :

1. H ow m any re act io n tu bes does Sara n eed to p re pare in th is exp erim ent?

2 . W hat sh ould e ach r e act io n tu be co nta in ?

3. F ollo w th e e xa mple in th e le ft p anel o f F ig ure 4 b elo w to co mple te th e g el p ict ure in th e r ig ht p anel.

a nd u plo ad w hat it w ould lo ok like ) T he D NA b ands sh ould co rre sp ond to th e fr a gm ent si ze s sh ow n o n th e le ft si de o f

th e p anel a nd r e fle ct th e r e su lts fr o m th e a bse nce o f d dA TP in o ne o f th e r e act io ns. Fig ure 4 . G el im age fr o m E xp erim ent 2 . U se th e le ft p anel a s an e xa mple fo r fillin g in th e r ig ht p anel.

a “ ? ” m ark to in dica te it.