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July 15 – 22, 2007
第 18 届国际生物奥林匹亚

PRACTICAL EXAM 4 实作4 GENETICS 遗传学 【会先进行色盲检验】 TASK A. Sequence confirmation of a cDNA 23 marks 一段 cDNA 序列的组成 (20 分) TASK B. Genetics of coat colour in dogs 16 marks TASK C. Genetic control of seed coat colour and seed shape in beans 20 marks 豆子种皮颜色及种子性状遗传的控制 (20分) Time allowed: 90 minutes 共90分钟 WRITE ALL ANSWERS IN THIS EXAM BOOKLET. 所有答案要写在这本试卷上 WRITE YOUR 4-DIGIT STUDENT CODE IN THE BOX BELOW AND ON THE TOP OF EACH PAGE OF THIS BOOKLET 将你的 4 码学生代码写在下栏中并写在其它各页的上方 STUDENT CODE 学生代码

STUDENT CODE _____________________
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TASK A. Sequence Confirmation of a cDNA (23 marks)
一段cDNA序列的组成(23分) Objective: To isolate plasmid DNA containing a cDNA of interest and to determine the sequence of the cDNA. 目的:分离一段含有待研究之 cDNA 的质体 DNA,并决定此 cDNA 的序列 Introduction: To over-express a gene of interest in a plant or animal you must first isolate the gene of interest in the form of a cDNA. You have done this and in order to amplify this DNA, you have cloned it into the pBluescript SK plasmid vector which you have subsequently used to transform bacteria cells. You must now carry out a quick plasmid preparation to isolate the plasmid and confirm the sequence of your cDNA insert. 介绍:要过度表现植物或动物有待研究的一个基因,你必须先以 cDNA 的形式 分离出这段基因;若你已完成分离,要放大这段 DNA 并 clone 在 pBluescript SK 质体的载体中,接着用这载体来转形细菌;你必须快速进行质体制备,以分离质 体并确定此 cDNA 插入段的序列。 Materials 材料Quantity (数)量 ? Bacterial cell culture 4 mL 细菌培养液 ? 1.5 mL microcentrifuge tubes 5 1.5 mL 微离心管 ? Microcentrifuge rack 1 微离心管架 ? P1000 micropipettor 1 P1000微吸管 ? Box of 200-1000 uL pipette tips 1 200-1000 uL吸管尖盒 ? GET buffer (1.5 mL tube) 0.5 mL GET缓冲液(1.5 mL小离心管) ? 10% Sodium Dodecyl Sulphate (1.5 mL tube) 0.5 mL 10% SDS (1.5 mL小离心管) ? 2 N NaOH (1.5 mL tube) 0.5 mL 2 N NaOH (1.5 mL 小离心管) ? 3 M Potassium 5 M Acetate (1.5 mL tube) 0.5 mL 3 M 钾 5 M 醋酸 (1.5 mL小离心管) ? 95% ethanol (Falcon tube) 3 mL 95% 乙醇 (试管)
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? Distilled water (Falcon tube) 3 mL 蒸馏水 (试管) ? Timer 1 定时器 ? Tube labels 2 试管标签 ? Marker pen 1 签字笔 ? Red card 1 红卡片 ? Garbage (tips & tubes) bag 1 垃圾袋 ? Access to a microcentrifuge 微离心机 ? Access to vortex 震荡机 NOTE: Before beginning this task, be sure that you have all the materials listed above. If you do not, raise your RED card to call a lab assistant. 注意:开始动手前,先确定你有上述所有材料,若有缺少,举起红卡片请监考人 员来帮忙 Procedure 步骤 1. Pipette 1.5 mL of bacterial culture into each of two 1.5 mL microcentrifuge tubes. 两支 1.5 mL 微离心管中各加入 1.5 mL细菌培养液 2. Centrifuge the tubes in a benchtop microcentrifuge for 1 minute - make sure that the centrifuge rotor is BALANCED. 用微离心机离心1分钟,离心前要确定离心转子(rotor)的平衡 3. Completely remove and discard back into the overnight tube the growth medium from each tube. 将两管中的上清液完全移除 4. Add 100 uL of GET (Glucose-EDTA-Tris) buffer pH 7.9 to the cell pellet (no need to cap the tubes) - vortex vigorously to resuspend the pellet and leave at room temperature for 5 minutes. 在细胞沈淀上加入100 uL pH 7.9 的GET (Glucose-EDTA-Tris)缓冲液(不需盖 上盖子),激烈震荡5分钟以悬浮细胞的沈淀,置于室温5分钟 5. In a separate 1.5 mL microcentrifuge tube, make a combined mixture of 1% SDS and 0.2 N NaOH in water to a final volume of 1 mL. 在另一支1.5 mL微离心管中,混合 1% SDS 及0.2 N NaOH到水中,使最后体
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积成为1 mL. 6. To each tube from 4. above add 200 uL of this freshly prepared mixture of 1% SDS and 0.2 N NaOH - cap the tubes and invert 4-5 times. 在上述步骤4的每支1.5 mL微离心管中,加入新配的200 uL1% SDS及0.2 N NaOH混合液,盖上盖子并倒置、正置4-5次 7. Incubate at room temperature for 3 minutes. 在室温培养3分钟 8. To each tube add 150 uL 5M KOAc (3 M potassium and 5 M acetate), cap the tubes and shake briefly by hand to mix. 在每支小管中加入150 uL 5M KOAc (3 M 钾与 5 M 醋酸) ,盖上盖子用手摇混 合 9. Incubate at room temperature for 3 minutes. 在室温培养3分钟 10. Centrifuge the tubes for 3 minutes - full speed in microcentrifuge remember to balance the rotor. 用微离心机全速离心3分钟 – 记得离心前要先平衡 11. Label 2 clean microcentrifuge tubes with your 4-digit student code number. 将你的4码学生代码标记在干净的2支微离心管上 12. Pipette the supernatant from each of the centrifuged tubes into each of the clean tubes. Discard the original tube which now contains a white pellet - this is bacterial chromosomal DNA. 将每支微离心管中的上清液吸出,置于新的微离心管中,弃置原管,该管中的白 色沈淀即为细菌染色体DNA 13. Add 800 uL of 95% ethanol to each tube. Cap the tubes, shake vigorously by hand for 10 sec and leave on the bench for 10 minutes. 在各管中加入800 uL 95%乙醇,盖上盖子用手摇混合10秒,静置10分钟 14. Centrifuge the tubes for 5 minutes - full speed in microcentrifuge. 用微离心机全速离心5分钟 15. Pour off the supernatant from each tube, cap the tube and raise your RED card. 倒出各管中的上清液,盖上盖子并举起你的红卡片 16. The lab assistant will check your pellet (10 marks for a white pellet). 让监考人员检查你的沈淀 (白色沈淀得10分)
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17. The lab assistant will then give you the sequence trace for your plasmid and cDNA. The cDNA was sequenced from the T promoter.

监考人员会给你质体及cDNA序列标记,此cDNA将由T 的起动子开始定序

18. Check your sequence (starting at nucleotide 21) against that for the pBluescript vector and answer the questions on page 5. 以pBluescript载体(由第21核苷酸起)检视你的序列,并回答第5页的问题 PLASMID MAP AND MULTIPLE CLONING SITE SEQUENCE FOR pBLUESCRIPT pBLUESCRIPT 的质体舆图及多选殖位(multiple cloning site)序列

Questions (13 marks) 问题(13 分) 1. The enzyme site into which you cloned your fragment of DNA is __________.
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用以选殖你DNA片段序列的酵素切位为何 NOTE: The first letter of the enzyme?s name is located above the first nucleotide of its recognition sequence. (5 marks). 注意:酵素名称的第一字母是在其所辨识序列的第一个核苷酸上面 (5 分) 2. List the first 20 nucleotides of your fragment of DNA (not including the restriction site sequence). (2 marks) 写出你那段DNA最前面的20个核苷酸的序列 (不包括限制位的序列) (2 分)
1 Nucleotide 核苷酸 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

3. Find the first start codon. Using the genetic code table provided, and starting with the start codon, translate the first 21 nucleotides into their appropriate amino acids. (4 marks) 找出第一个起始密码子, 用所提供的遗传密码表,由起始密码子开始将头21个核 苷酸译成对应的氨基酸序列 (4 分) Start codon
Amino acid 氨基酸 1 Nucleotide 核苷酸 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

4. (a) If the nucleotide at position 13 was mutated to an ?A?, what would be the corresponding amino acid? (1 mark) 如果核苷酸第 13 个位置突变成为 “A”,将会对应成为何种氨基酸 (1 分)

(b). If the nucleotide at position 14 was mutated to an ‘A’, what would be the corresponding amino acid? (1 mark) 如果核苷酸第 14 个位置突变成为 “A”,将会对应成为何种胺基酸 (1 分)

This table shows the 64 codons and the amino acid each codon codes for. The direction is 5' to 3'.
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本表显示 64 个密码子及其所对应的氨基酸,方向是 5' to 3'

2nd base
UUU(Phe/F)Phenylala nine UUC(Phe/F)Phenylala

UCU(Ser/S)Serine UCC(Ser/S)Serine UCA(Ser/S)Serine UCG(Ser/S)Serine

UAU(Tyr/Y)Tyrosine UAC(Tyr/Y)Tyrosine UAAOchre(Stop) UAG Amber (Stop) e


UGC(Cys/C)Cystein e UGA Opal(Stop)


nine UUA (Leu/L)Leucine UUG (Leu/L)Leucine

UGG(Trp/W)Tryptop han

CUU (Leu/L)Leucine CUC(Leu/L)Leucine

CCU(Pro/P)Proline CCC(Pro/P)Proline CCA(Pro/P)Proline CCG(Pro/P)Proline

CAU(His/H)Histidine CAC(His/H)Histidine CAA(Gln/Q)Glutami ne CAG(Gln/Q)Glutami ne

CGU(Arg/R)Arginine CGC(Arg/R)Arginine CGA(Arg/R)Arginine CGG(Arg/R)Arginine


CUA (Leu/L)Leucine CUG (Leu/L)Leucine

1st bas e

AUU (Ile/I)Isoleucine AUC (Ile/I)Isoleucine AUA (Ile/I)Isoleucine AUG (Met/M)Methionine

ACU(Thr/T)Threon ine ACC(Thr/T)Threon ine ACA (Thr/T)Threonine ACG (Thr/T)Threonine

AAU(Asn/N)Aspara gine AAC(Asn/N)Aspara gine AAA(Lys/K)Lysine AAG (Lys/K)Lysine

AGU (Ser/S)Serine AGC(Ser/S)Serine AGA(Arg/R)Arginine AGG(Arg/R)Arginine

GUU (Val/V)Valine GUC (Val/V)Valine GUA (Val/V)Valine

GCU(Ala/A)Alanin e GCC(Ala/A)Alanin e GCA(Ala/A)Alanin e GCG(Ala/A)Alanin e

GAU(Asp/D)Asparti c acid GAC(Asp/D)Asparti c acid GAA(Glu/E)Glutami c acid GAG(Glu/E)Glutami c acid

GGU(Gly/G)Glycine GGC(Gly/G)Glycine GGA(Gly/G)Glycine GGG(Gly/G)Glycine

G GUG (Val/V)Valine

Task B. 删除 Task C. Genetic Control of Seed Coat Colour and Seed Shape in Beans (20 points) 豆子种皮颜色及种子性状遗传的控制 (20 分) Material 材料 ? 1 plastic bag containing flat red parent beans – Do not open 1塑料袋装有扁平红色的亲代豆子 – 不可打开 ? 1 plastic bag containing round red parent beans – Do not open
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1塑料袋装有圆厚红色的亲代豆子 – 不可打开 ? 1 plastic bag containing F seeds (flat yellow) from the cross between the

parent beans – Do not open 1塑料袋装有上述亲代交配后所产生的第一子代豆子(扁平黄色) – 不可打开 ? 1 plastic bag of F bean seeds (representing 250 F plants) – This bag may
2 2

be opened 1塑料袋装有用以代表250个第二子代植株每株一颗的豆子 – 此袋可以打 开 To help you answer the questions below, fill in the following table: 为了帮助你回答下列问题,请填写下列空格
Generation 世代 Parent 1 (亲代 1) Parent 2 (亲代 2) F from a cross between

Seed shape 种子形状 (round or flat) (圆厚或扁平)

Seed coat colour 种皮颜色 (yellow or red) (黄色或红色)

these two parents (第一子 代,来自于上述亲代互交)

Answer the following questions. 回答下列问题 1. Is the seed coat colour controlled by (circle one) 种皮颜色受到何种方式调控(圈选一个答案) (i) one gene 单一基因 (ii) more than one gene? (1 mark) 超过一个基因 (1分) 2. a) Red seed coat colour is (circle one) 红色种皮是何种性状(圈选一个答案) (i) dominant 显性 (ii) partially dominant 部份显性 (iii) recessive (1 mark) 隐性 b) Round seed shape is (circle one)
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圆厚种子是何种性状(圈选一个答案) (i) dominant 显性 (ii) partially dominant 部份显性 (iii) recessive (1 mark) 隐性 (1分) 3. (a) There are four phenotypes in your sample of F seeds. Classify the seeds

into these phenotypic classes and write the number of each phenotype in the table below. (2 marks) F 种子有四种外表型,将这些种子依外表型分开并将每种的数目写在下表中 (2

Number of seeds 种子数 Phenotype 外表型 (seed colour/ seed shape) (种子颜色/种子形状) round, red 圆厚红色 flat, red 扁平红色 round, yellow圆厚黄色 flat, yellow扁平黄色 Total总数 (= number of F plants)

(= F 植株数目)

Use these F2 segregation data to answer the following questions: 用这些F 分离的资料回答下列问题:

4. (a) From your data how many genes could be controlling seed shape? (1 mark) 从你的数据来看,多少基因可能控制种子形状? (1分) (b) How many round beans and how many flat ones would you expect in a population this size? 在这样大小的族群中,你期待会有多少圆厚的豆子?多少扁平的豆子? ROUND ______________ FLAT __________ (2 marks) 圆厚 扁平 (2分) (c) Is this segregation ratio significantly different from the observed ratio (circle one)? 这分离的比率与观察到的比率差别是否有意义 (圈选一个答案) YES NO (1 mark) 是 否 (1分)
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And what is the probability? 机率是多少?

(3 marks) (3分)

5. (a) From your data how many genes could be controlling seed coat color? ______ (1 mark) 从你的数据来看,多少基因可控制种皮的颜色? (1分) (b) How many red beans and how many yellow beans would you expect in a population this size? 在这样大小的族群中,你期待会有多少红色的豆子?多少黄色的豆子? RED ______________ YELLOW __________ (3 marks) 红色 黄色 (3分) (c) Is this segregation ratio significantly different from the observed ratio? (circle one) 这分离的比率与观察到的比率差别是否有意义 (圈选一个答案) YES NO (1 mark) 是 否 (1分) And what is the probability? (3 marks) 机率是多少? (3分)
Chi-square Distribution 卡方分布 Probability 机率 df

1 2 3 4

0.95 0.004 0.10 0.35 0.71

0.90 0.02 0.21 0.58 1.06

0.80 0.06 0.45 1.01 1.65

0.70 0.15 0.71 1.42 2.20

0.50 0.46 1.39 2.37 3.36

0.30 1.07 2.41 3.66 4.88

0.20 1.64 3.22 4.64 5.99

0.10 2.71 4.60 6.25 7.78

0.05 3.84 5.99 7.82 9.49

0.01 6.64 9.21 11.34 13.28

0.001 10.83 13.82 16.27 18.47


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