Restriction mapping and gene analysis

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Experimental Section 0307-4412(95)00158-1 Restriction M a p p i n g and Gene Analysis M SIMSEK Department of Biochemistry College of Medicine Sultan Qaboos University PO Box 35, AI Khoud Postal Code 123, Muscat-Oman Introduction This short experiment can be completed in a two-hour laboratory session. It is composed of two parts: (i) restriction mapping of a plasmid DNA, and (ii) analysis of a provided DNA sequence for a particular gene. In the first part, each student pair cuts a plasmid DNA with one or more restriction endonucleases and loads their sample onto one of the lanes of an agarose gel. During the electrophoresis period (1 h), each student is asked to locate a particular gene in the sequence of a given double stranded DNA using the partial N-terminal amino acid sequence of the encoded protein as a hint. At the end of the practical session, each student receives a photocopy of the gel electrophoresis data for restriction mapping. Although this laboratory exercise appears to be simple, it provides an opportunity for students to practice and integrate many concepts they have learned in a molecular genetics course.

Aim of the laboratory practical The restriction mapping experiment enables students to understand: (i) the specificity of restriction endonucleases, (ii) the separation of DNA fragments according to size by electrophoresis, (iii) the principles involved in the detection and visualization of DNA fragments by staining with ethidium bromide, and (iv) the difference in the electrophoretic mobility of supercoiled plasmid DNA versus its linearized or relaxed circular forms. 1The second part of the experiment enables each student to: (i) use the Genetic Code table to locate a desired gene among the many other open reading frames (ORF) encountered, (ii) recognize the degeneracy involved in codon utilization, (iii) determine the total length of a gene and its encoded protein product, and finally (iv) locate the Shine-Dalgarno sequence 2 in the DNA sequence of a bacterial gene. In this experiment, the DNA sequence provided contained the ampicillin resistance gene (AmpR) which codes for ampicillinase with a signal sequence. Therefore, the students also refresh and integrate their knowledge about the properties and function of signal sequences.

Materials Plasmid pBR322 and DNA size standards (1 kbp ladder) were purchased from Gibco BRL Life Technologies, Inc. Restriction endonucleases (EcoRl, Sail and Pstl) and One-Phor-All-Buffer (OPA, 10 × concentrated) were from Pharmacia, the latter contained 100 mM Tris-acetate pH 7.5, 100 mM magnesium acetate, and 500 mM potassium acetate. Electrophoresis grade agarose was from Bio-Rad Laboratories. The buffer for agarose gel electrophoresis TAE (50 x concentrated) was prepared using reagent grade chemicals and contained 2 M Tris-acetate pH 8.0, and 50 mM EDTA. The loading gel buffer contained 60% glycerol, 50 mM EDTA pH 8.0, and 0.125% bromophenol blue (Bio-Rad). A stock solution of ethidium bromide (Sigma) was prepared in distilled water (1 mg/ml). CAUTION: Ethidium bromide is a carcinogen. It must be handled with care using gloves when preparing gels or buffers. Solutions contaminated

BIOCHEMICAL EDUCATION 24(2) 1996

Table 1 Protocol for the cutting of plasmid DNA with different restriction enzymes Tube no.

Plasmid DNA (pl)

Distilled water (ld)

Restriction enzyme (id)

1, 2 3, 4 5, 6 7, 8 9, 10 11, 12 13, 14

5 5 5 5 5 5 5

15 10 10 10 5 5 5

0 5 PstI 5 EcoRI 5 Sail 5 PstI+5 EcoRI 5 PstI+5 Sail 5 EcoRI+5 Sail

The concentration of plasmid DNA was adjusted to 0.05 pg/pl and contained the OPA buffer with magnesium ions at six-foldconcentrated form. Each restrictions enzyme was diluted to 0.5 units per/~1 with the OPA buffer (final strength 1 x ) 10 minutes before the practical session.

with ethidium bromide should be disposed of safely according to Maniatis et a l . 3

Methods Cutting plasmid DNA with three different restriction endonucleases Students work in pairs to perform one or more of the reactions shown in Table 1 by mixing the indicated volumes of plasmid DNA, restriction enzymes and distilled water in a 1.5 ml Eppendorf tube. All samples are incubated at 37°C for 30 minutes in a water bath, after which the reactions are stopped by adding 4/~1 of the loading gel buffer to each sample.

Agarose gel electrophoresis

A wide mini sub-cell unit (Bio-Rad) is used for electrophoresis. Agarose gels (1% each) are prepared by solubilizing the agarose in 1.0 x TAE buffer containing 0.5/~g ethidium bromide per ml. The gels are cast by pouring the molten agarose (after cooling to about 60°C) onto gel trays taped at both ends, and combs are inserted to form the wells. After the gels have solidified (30 minutes), the combs and tapes are removed and the gel trays are placed in Bio-Rad tanks containing 0.5 x TAE buffer and ethidium bromide (0.5 pg/ml). Electrophoresis of the gel is performed at 90 volts for one hour. At the end, a polaroid photograph of the gel is taken by the intructor using a PM4 polaroid camera set (Sigma) and a UV-transilluminator (Pharmacia). A photocopy of the picture is given to each student for analysis.

DNA sequence analysis

A software program (Hitachi Hibio Dnasis) was used to access DNA sequences stored on a CD-ROM (gene bank). A 1200 bp partial sequence of pBR322 containing the AmpR gene with its flanking sequences was generated in two different orientations by using the sequence editing program of Dnasis. If such software is not available, it may be possible to generate similar sequences by photocopying desired regions of published DNA sequences in the literature.

Results Agarose gel electrophoresis and restriction mapping Figure 1 shows the electrophoretic pattern of DNA fragments obtained by students in one of the practical sessions after cutting pBR 322 with one or more restriction enzymes. Each student is expected to estimate the length of pBR322 (in base-pairs) based on their experimental data and also to determine the relative cutting positions of the three enzymes used (PstI, EcoRI, and Sail) on a circular map. The actual size of pBR322 is 4361 bp/Figure 2 shows a linear map of this plasmid with the positions of the three enzymes used. Students are expected to calculate the distances in base pairs between the restriction sites of these enzymes and

118

Table 2 Questions fi)r further stud)' hn

M

1

2

3

4

5

7 8

9

10 11 12 13 14

Figure 1 Electrophoretic pattern of DNA fragments produced after cutting pBR322 with different restriction endonucleases. Electrophoresis was in a 1% agarose gel for 1 hour at 90 ~: Lanes: M, 1 kbp ladder as DNA size-standards; 1, 2, untreated pBR322; 3, 4, PstI; 5, EcoR k 7, 8, Sal I; 9, 10, Pst I + E c o R I; 11, 12, Pst I + Sal k 13, 14, EcoR I + Sal I. Sample 6 (EcoR I treatedpBR322) was a duplicate of that in lane 5 but was lost before electrophoresis to compare their findings with the experimental values estimated from the relevant lanes in Figure 1. This allows students to learn about the resolving power of 1% agarose gel for the separation of different size D N A fragments. For example, they should realize that the data in Figure 1 (lanes 4-9) may be used to estimate the size of pBR322 between 4 and 5 kbp with an uncertainty factor about 0.5 kbp. However, a better estimate may be made from the double digests (Figure 1, lanes 9-14) in which two smaller D N A fragments were produced and their size can be estimated more accurately ( + 100 to 200 bp). Figure 1 provides a negative control (lanes 1 and 2) where the plasmid DNA was not treated with any restriction endonucleases. There were two bands observed for the untreated pBR322. The fast moving band contained the supercoiled form of plasmid D N A and the slower one was due to the circular relaxed form/ There was no detectable linear form in this plasmid preparation. Students are expected to note the difference in the electrophoretic mobility of the different forms of the same plasmid DNA and to offer an explanation as to why the supercoiled form has a higher mobility than either the linear form (lanes 4-9) or the circular relaxed form (lanes 1 and 2).

Gene analysis The D N A sequence provided for gene analysis is shown in Figure 3 in two different orientations. Each sequence (1200 bp) contains the E coli ampicillinase gene with some flanking sequences. The ampicillinase is a targeted periplasmic protein. Its partial N-terminal sequence (after the removal of a 15 amino acid signal peptide) is as follows? A l a - P h e - C y s - L e u Pro-Val-PheEach student works independently to locate the gene using a copy of the Genetic Code and the partial N-terminal sequence of the protein. Most students are able to locate the desired gene in less than 30 minutes. Once the gene is located with its initiation and termination codons, the entire length of the gene could be determined as 861 bp coding for a protein that contains 286 amino acids including the signal peptide sequence. EcoRI I

0

Sill

P~I

I

I

650

3612

Figure 2 Linear map of pBR322 BIOCHEMICAL EDUCATION 24(2) 1996

EcoRI t

4361

(1) How can you explain the difference in the electrophoretic mobility of pBR322 molecules in lanes 2 and 3 of Fig l? Can you use the linear DNA size markers provided in Fig 1 (lane M) to estimate the size of DNA molecules in lane 2 of the same Figure? (2) What are the principles involved in the visualization of DNA molecules by ethidium bromide? Assuming that the lowest limit of detection by ethidium bromide is around 10 ng DNA per band, what will be minimum number of 1000 bp-long DNA fragments that can be detected in a band by ethidium bromide? (3) How can you explain the difference in the intensity of two DNA bands produced in Fig 1 (lanes 9 or 13)? (4) What is the difference between a complete versus a partial digestion of DNA with a given enzyme? Which of the following lanes in Fig 1 (7, 8, 9, 11, 12, and 13) contain incomplete digestion? (5) What is the function of the Shine-Dalgarno sequence found in most bacterial mRNAs? Can you locate this sequence in Fig 3? (6) What will be effect of deleting one base pair from the DNA sequence in Fig 3a at the following positions: (a) 30, (b) 2{12, (c) 210, (d) 510 and (el 1190? (7) What is the function of leader peptide for ampicillinase? Determine the percentage of hydrophobic amino acids in this sequence (Fig 3). (8) The mature ampicillinase will be composed of 271 amino acids. Would you expect the number of two different amino acids (eg methionine and alanine) in this protein to be the same or not'? Explain your answer.

Discussion The first part of this practical covered restriction digestion of a plasmid DNA with various enzymes and separation of the resulting DNA fragments by electrophoresis on an agarose gel. Students calculate the size of expected fragments from a linear map of pBR322 (Figure 2) and compare these with the experimental values deduced from Figure 1. In our class there were 28 students in cach practical session forming 14 pairs and each pair performed one of the reactions in Table 1. All the digests were loaded onto a single gel to generate a result to be shared by all student groups. This approach is useful if saving is required for many items including plasmid DNA, restriction endonucleases, the components for agarose gel electrophoresis and polaroid films. Alternatively, a student pair can perform all the reactions in Table 1 independently and generate their own results. In such a case, there would possibly be no need to perform the reactions in duplicate since a mistake in a given reaction will not affect the results for the entire class. By avoiding duplications one can also increase the number of restriction enzymes or their combinations in Table 1. Usually the restriction digestion of DNA is carried out in 10-20/A by pipetting small (0.5-1 /11) volumes of enzymes and 10 x concentrated buffer. Pipetting of small volumes, especially by students, may introduce unnecessary errors which was avoided by diluting the enzymes and incorporating the OPA buffer into plasmid DNA solution (Table 1). In a pre-run experiment the amount of restriction enzymes and incubation time (30 min at 37°C) were found to be satisfactory for complete digestion of the plasmid D N A used (0.25 #g). Student results, however, showed some incomplete digestions in a few incubation mixtures (Figure 1, lanes 8, 11, and 12). These were most likely due to errors in pipetting or mixing of the reactants properly. Nevertheless, the presence of some additional DNA bands due to incomplete digestion did not interfere with the interpretation of data and mapping of restriction sites.

119 B

A I0 51 T T C T T G A A G A 3 l AAGAACTTCT 70 A A ~ TTACCAAAGA 130 TTTA'x-~-xTL'C AAATAAAAAG 190 GCTTCAATAA CGAAGTTATT 250 TCC~.-~-~-X-XTX" AGGGAAAAAA 310 AAAAGATGCT TTTTCTACGA 370 CGGTAAGATC GCCATTCTAG 430 AGTTCTGCTA TCAAGACGAT 490 - CCGCATACAC GGCGTATGTG 550 TA~.,GATGGC ATGCCTACCG 610 TGCGGCCAAC ACGCCGGTTG 670 CAACATGGGG GTTGTACCCC 730 ACCAAACGAC TGGTTTGCTG 790 ATTAACTGGC TAATTGACCG 850 GGATAAAGTT CCTATTTCAA 910 TAAATCTGGA ATTTAGACCT 970 TAAGCCCTCC ATTCGGGAGG 1030 AAATAGACAG TTTATCTGTC 1090 AGTTTACTCA TCAAATGAGT 1150 GGTGAAGATC CCACTTCTAG

20 30 40 50 C G A A A G G G C C T C G T G A T A C G CCTA'FI"F#£A T A G G T T A A T G GCTTTCCCGG AGCACTATGC GGATAAAAAT ATCCAATTAC 80 90 i00 110 TAGACGTCAG GTGGCACTTT TCGGGGAAAT GTGCGCGGAA ATCTGCAGTC CACCGTGAAA AGCCCCTTTA CACGCGCCTT 140 150 160 170 TAAATACATT C~AATATGTA TCCGCTCATG AGACAATAAC ATTTATGTAA GTTTATACAT AGGCGAGTAC TCTGTTATTG 230 200 22o TATTGAAAAA GGAAGAG GTATTCAA CATTTCCGTG ATAA~-F~-x-x-s" ~ CATAAGTT GTAAAGGCAC 290 260 270 280 GCGGCA'FI-~-~" G C C T T C C T G T "F~TL~JCTCAC C C A G A A A C G C G G T C T T T G CG CGCCGTAAAA CGGAAGGACA AAAACGAGTG 350 320 330 340 GAAGATCAGT TGGGTGCACG AGTGGGTTAC A T C G A A C T G G CTTCTAGTCA ACCCACGTGC TCACCCAATG T A G C T T G A C C 410 380 390 400 C T T G A G A G T T T T C G C C C C G A AGAACG'A'FP~ C C A A T G A T G A GAACTCTCAA AAGCGGGGCT TCTTGCAAAA GGTTACTACT 470 440 450 460 TGTGGCGCGG TATTATCCCG TGTTGACGCC GGGCAAGAGC ACACCGCGCC ATAATAGGGC ACAACTGCGG CCCGTTCTCG 530 500 510 520 TATTCTCAGA ATGACTTGGT TGAGTACTCA CCAGTCACAG ATAAGAGTCT TACTGAACCA ACTCATGAGT GGTCAGTGTC 590 560 570 580 ATGACAGTAA GAGAATTATG CAGTGCTGCC ATAACCATGA TACTGTCATT CTCTTAATAC GTCACGACGG TATTGGTACT 650 620 630 640 TTACTTCTGA CAACGATCGG AGGACCGAAG GAGCTAACCG AATGAAGACT GTTGCTAGCC TCCTGGCTTC C T C G A T T G G C 710 680 690 700 GATCATGTAA CTCGCCTTGA TCGTTGGGAA CCGGAGCTGA CTAGTACATT GAGCGGAACT AGCAACCCTT GGCCTCGACT 770 740 750 760 GAGCGTGACA CCACGATGCC TGCAGCAATG GCAACAACGT CTCGCACTGT GGTGCTACGG ACGTCGTTAC CGTTGTTGCA 830 800 810 820 GAACTACTTA CTCTAGCTTC CCGGCAACAA TTAATAGACT CTTGATGAAT GAGATCGAAG GGCCGTTGTT AATTATCTGA 890 860 870 880 GCAGGACCAC TTCTGCGCTC GGCCCTTCCG GCTGGCTGGT CGTCCTGGTG AAGACGCGAG CCGGGAAGGC CGACCGACCA 950 920 930 840 GCCGGTGAGC GTGGGTCTCG CGGTATCATT GCAGCACTGG CGGCCACTCG CACCCAGAGC GCCATAGTAA CGTCGTGACC 980 990 1000 i010 CGTATCGTAG TTATCTACAC GACGGGGAGT C A G G C A A C T A GCATAGCATC AATAGATGTG CTGCCCCTCA GTCCGTTGAT 1040 1050 1060 1070 ATCGCTGAGA TAGGTGCCTC ACTGATTAAG C A ~ T A G C G A C T C T A T C C A C G G A G TGACTAATTC G T A A C C A T T G ii00 Iii0 1120 1130 TATATACTTT AGATTGATTT AAAACTTCAT TTTTAATTTA A T A T A T G A A A T C T A A C T A A A "~-~TA~AAGTA A A A A T T A A A T 1160 1170 1180 1190 ~-~'i'x'x'I~ATAA T C T C A T G A C C A A A A T C C C T T A A C G T G A G T G A A A A A C T A T T A G A G T A C T G G-ITI-£AGGGA A T T G C A C T C A

60 TCATGATAAT AGTACTATTA 120 CCCCTATTTG GGGGATAAAC 180 CCTGATAAAT GGACTATTTA 240 TCGCCCTTAT AGCGGGAATA 300 TGGTGAAAGT A C C A ~-x'I"I'CA 360 ATCTCAACAG TAGAGTTGTC 420 GCA~"x-x-x-~'AA CGTGAAAATT 480 AACTCGGTCG TTGAGCCAGC 540 AAAAGCATCT TTTTCGTAGA 600 GTGATAACAC CACTATTGTG 560 C~'Iu.-~,-i--i~CA GAAAAAACGT 720 ATGAAGCCAT TACTTCGGTA 780 TGCGCAAACT ACGCGTTTGA 840 GGATGGAGGC CCTACCTCCG 900 TTATTGCTGA AATAACGACT 960 GGCCAGATGG CCGGTCTACC 1020 TGGATGAACG ACCTACTTGC 1080 TGTCAGACCA ACAGTCTGGT 1140 AAAGGATCTA TTTCCTAGAT 1200 TTTCGTTCCA 3 t A A A G C A A G G T 51

10 20 30 40 5 I TGGAACGAAA ACTCACGTTA AGGGATTTTG GTCATGAGAT 3' A C C T T G C T T T T G A G T G C A A T T C C C T A A A A C C A G T A C T C T A 70 80 90 100 TAGATCCTTT TAAATTAAAA ATGAAGTTTT AAATCAATCT ATCTAGGAAA ATTTAATTTT TACTTCAAAA TTTAGTTAGA 130 140 150 160 TGGTCTGACA GTTACCAATG CTTAATCAGT GAGGCACCTA ACCAGACTGT ~GGTTAC GAATTAGTCA CTCCGTGGAT 200 210 220 190 CGTTCATCCA TAGTTGCCTG ACTCCCCGTC GTGTAGATAA GCAAGTAGGT ATCAACGGAC TGAGGGGCAG CACATCTATT 250 260 270 280 CCATCTGGCC CCAGTGCTGC AATGATACCG CGAGACCCAC GGTAGACCGG GGTCACGACG TTACTATGGC GCTCTGGGTG 310 320 330 340 TCAGCAATAA ACCAGCCAGC CGGAAGGGCC GAGCGCAGAA AGTCGTTATT TGGTCGGTCG GCCTTCCCGG CTCGCGTCTT 380 390 400 370 GCCTCCATCC AGTCTATTAA TTGTTGCCGG GAAGCTAGAG CGGAGGTAGG TCAGATAATT AACAACGGCC CTTCGATCTC 440 450 460 430 AGTTTGCGCA ACGTTGTTGC CATTGCTGCA GGCATCGTGG TCAAACGCGT TGCAACAACG GTAACGACGT CCGTAGCACC 500 510 520 490 ATGGCTTCAT TCAGCTCCGG TTCCCAACGA TCAAGGCGAG TACCGAAGTA AGTCGAGGCC AAGGGTTGCT AGTTCCGCTC 550 560 570 580 TGCAAAAAAG CGGTTAGCTC CTTCGGTCCT CCGATCGTTG A C G~q-i-i-i'~C G C C A A T C G A G G A A G C C A G G A G G C T A G C A A C 610 620 630 640 G~I'ATCAC TCATGGTTAT GGCAGCACTG CATAATTCTC CA~,AT&G'I~ AGTACCAATA CCGTCGTGAC GTATTAAGAG 670 680 690 700 AGATG~-X-I~-I• C T G T G A C T G G T G A G T A C T C A A C C A A G T C A T TCTACGAAAA GACACTGACC ACTCATGAGT TGGTTCAGTA 730 740 750 760 CGACCGAGTT GCTCTTGCCC GGCGTCAACA CGGGATAATA GCTGGCTCAA CGAGAACGGG CCGCAGTTGT GCCCTATTAT 790 800 810 820 TTAAAAGTGC TCATCATTGG AAAACGTTCT TCGGGGCGAA A A T T T T C A C G A G T A G T A A C C T T T T G C A A G A AGCCCCGCYF~ 850 860 870 880 CTGTTGAGAT CCAGTTCGAT GTAACCCACT CGTGCACCCA GACAACTCTA GGTCAAGCTA CATTGGGTGA GCACGTGGGT 910 920 930 940 A~-x'/'I'CACCA G C G T T T C T G G G T G A G A A A ACAGGAAGGC TGAAAGTGGT CGCAAAGACC CACTCGTTTT TGTCCTTCCG 970 980 990 1000 ATAAGGGCGA CACGGAAATG TTGAATACTC ATACTCTTCC TATTCCCGCT GTGCCTTTAC AACTTATG AGAAGG 1030 1040 10 1060 ATTTATCAGG GTTATTGTCT CATGAGCGGA TACATATTTG TAAATAGTCC CAATAAeAGA GTACTCGCCT ATGTATAAAC 1090 1100 III0 1120 CAAATAGGGG TTCCGCGCAC ATTTCCCCGA AAAGTGCCAC GTTTATCCCC AAGGCGCGTG TAAAGGGGCT TTTCACGGTG 1150 1160 1170 1180 ATTATCATGA CATTAACCTA TAAAAATAGG CGTATCACGA T~T~GTACT GTAATT~T'A~:~z_~..C-~CAT&~

50 60 TATCAAAAAG GATCTTCACC ATAG'Fx-x-~-x'C C T A G A A G T G G ii0 120 AAAGTATATA TGAGTAAACT TTTCATATAT ACTCATTTGA 170 180 TCTCAGCGAT CTGTCTATTT AGAGTCGCTA GACAGATAAA 230 240 CTACGATACG GGAGGGCTTA GATGCTATGC CCTCCCGAAT 250 300 GCTCACCGGC TCCAGATTTA CGAGTGGCCG AGGTCTAAAT 350 350 GTGGTCCTGC AACTTTATCC CACCAGGACG TTGAAATAGG 410 420 TAAGTAGTTC GCCAGTTAAT ATTCATCAAG CGGTCAATTA 470 480 TGTCACGCTC GTCGTTTGGT ACAGTGCGAG CAGCAAACCA 530 540 TTACATGATC CCCCATGTTG AATGTACTAG GGGGTACAAC 590 600 TCAGAAGTAA GTTGGCCGCA AGTCTTCATT CAACCGGCGT 650 660 TTACTGTCAT GCCATCCGTA AATGACAGTA CGGTAGGCAT 710 720 TCTGAGAATA GTGTATGCGG AGACTCTTAT CACATACGCC 770 780 CCGCGCCACA TAGCAGAACT GGCGCGGTGT ATCGTCTTGA 830 840 AACTCTCAAG GATCTTACCG TTGAGAGTTC CTAGAATGGC 890 900 A C T G A T C T T C AGCA~,'~'~'~'~" TGACTAGAAG TCGTAGAAAA 950 960 AAAATGCCGC AAAAAAGGGA T T T T A C G G C G "A'A'A'A'A'A'CCCT I010 1020 "~TITI"CAATA T T A T T G A A G C AAAAAGTTAT AATAACTTCG 1070 1080 AATGTATTTA GAAAAATAAA T T A C A T A A A T ~-I-XTL-I'ATTT 1130 1140 CTGACGTCTA AGAAACCATT GACTGCAGAT TCTTTGGTAA 1190 1200 GGCC~T~-~'CG T C T T C A A G A A 3 t CCGGGI~J~3C ~ G ~ G T T C ' ~ T 5~E'

Figure 3 Nucleotide sequenc eof the E coli ampicillin resistance gene in two different orientations (A & B). The sequence B corresponds to the nucleotide positions 3162 to 4361 in the published pBR322 sequence.4 The initiation (A TG ) and termination codons (TAA) for the AmpR gene are boxed and the Shine-Dalgarno sequence (GGAAG) is underlined in both sequences. In the second part of this practical, each student carried out an open reading frame (ORF) analysis on a provided 1200 bp D N A to locate a particular gene that contained 861 bp in its coding region. In addition to keeping students active during a one hour long electrophoresis period, the ORF analysis also helped them to reinforce some fundamental concepts they had learned in molecular biology or genetics courses. In ORF analysis, it is preferable to provide double-stranded D N A sequences so that students are challenged to identify the coding sense strand of the desired gene. To promote independent analysis, each student was given one of the two orientations of the same D N A sequence (Fig. 3a or b). During this exercise they realized that the ORF analysis can only be performed in the 5' to 3' direction on a given D N A chain, and that both D N A chains need to be analyzed separately until the coding strand of the gene under analysis is identified. The ORF analysis can be time-consuming if performed manually in all three possible reading frames along the entire length of a 1200 nucleotide long D N A chain assuming that one was lucky to start the analysis with the chain containing the coding strand of the gene. Accordingly students were given some directions on how to perform the analysis in a more practical way. They first located the ATG triplets within the 300 nucleotides from the 5' end of a chain without paying attention to which reading frame they may be located. Then they started the ORF analysis at each ATG until they found an ORF containing more

B I O C H E M I C A L E D U C A T I O N 24(2) 1996

than 15 triplets. Only then did they translate it and compare the coded amino acids with the provided N-terminal sequence of ampicillinase. Using the directions given most students were able to locate the correct ORF for the ampicillinase in less than 30 min, and completed the analysis to determine the entire length of the AmpR gene in the remaining time. To benefit more from this practical, the students may be given a list of further questions (Table 2) to help them focus on various important points in the practical class and to reinforce some of the fundamental concepts in molecular biology. Acknowledgements This work was supported in part by Departmental funds. The author wishes to thank Professor K A Gumaa and Dr N Al-Wardy for helpful discussions and critical reading of the manuscript. References 1 Birnboim, H C and Doly, J (1979) Nucleic Acid Res 7, 1513-1523 2 Shine, J and Dalgarno, L (1974) Proc Natl Acad Sci USA 71, 1342-1346 3 Sambrook, J, Fritsch, E F and Maniatis, T (1989) Molecular Cloning, second edition, Cold Spring Harbor Publications 4 Sutcliffe, J G (1979) Cold Spring Harbor Syrup Quant Biol 43, 77-90 5 Sutcliffe, J G (1978) Proc NatlAcad Sci USA 75, 3737-3741