1
00:00:07,340 --> 00:00:11,290
PROFESSOR: So, first step,
we need to cut our DNA.

2
00:00:16,340 --> 00:00:26,446
Step one, cut, which is going to
be DNA restriction enzymes.

3
00:00:35,980 --> 00:00:39,910
It turns out, quite remarkably,
that if I have a

4
00:00:39,910 --> 00:00:54,640
sequence of DNA, five prime A G
C T A G A A T T C T T A C C

5
00:00:54,640 --> 00:00:58,000
three prime, and we'll
come backwards

6
00:00:58,000 --> 00:00:59,250
filling in the sequence.

7
00:01:09,170 --> 00:01:17,170
It turns out that molecular
biologists are so cool that

8
00:01:17,170 --> 00:01:24,100
they invented a protein that is
able to recognize that six

9
00:01:24,100 --> 00:01:28,900
letter sequence, G-A-A-T-T-C.

10
00:01:28,900 --> 00:01:33,360
And it is able-- actually it's
G-A-A-T-T-C on this strand,

11
00:01:33,360 --> 00:01:35,190
but what is it coming back?

12
00:01:35,190 --> 00:01:39,170
It's G-A-A-T-T-C. It's
the same thing.

13
00:01:39,170 --> 00:01:41,300
So actually this is
a palindrome.

14
00:01:41,300 --> 00:01:41,970
That's kind of nice.

15
00:01:41,970 --> 00:01:42,990
It's a palindrome--

16
00:01:42,990 --> 00:01:45,220
it's a reverse palindrome.

17
00:01:45,220 --> 00:01:50,530
It's the same word spelled
backwards on the other strand.

18
00:01:50,530 --> 00:01:56,530
So what it does is it
cuts it like that.

19
00:01:56,530 --> 00:02:04,340
And what it produces is a DNA
molecule like this and a DNA

20
00:02:04,340 --> 00:02:08,889
molecule like that, that's
mostly double stranded, but

21
00:02:08,889 --> 00:02:11,500
has a four base pair overhang.

22
00:02:11,500 --> 00:02:14,640
The overhang reads
T-T-A-A here.

23
00:02:14,640 --> 00:02:17,610
It reads A-A-T-T there.

24
00:02:17,610 --> 00:02:21,760
And remember this is five prime
to three prime, five

25
00:02:21,760 --> 00:02:24,720
prime to three prime.

26
00:02:24,720 --> 00:02:26,910
And there you go.

27
00:02:26,910 --> 00:02:31,030
This guy has its little
phosphate at the end there.

28
00:02:31,030 --> 00:02:35,156
This guy has his little
hydroxyl over there.

29
00:02:35,156 --> 00:02:36,406
And it cuts it.

30
00:02:38,770 --> 00:02:41,340
Now that is an incredible
piece of engineering.

31
00:02:41,340 --> 00:02:45,260
To come up with a protein, to
devise a protein, that is able

32
00:02:45,260 --> 00:02:48,850
to recognize those six bases
and cut at those six bases.

33
00:02:52,160 --> 00:02:53,970
And cut in just this
way making a really

34
00:02:53,970 --> 00:02:57,350
clean overhang here.

35
00:02:57,350 --> 00:03:00,850
It's this cool five
prime overhang.

36
00:03:00,850 --> 00:03:04,080
Who do you think invented
this cool protein?

37
00:03:04,080 --> 00:03:06,600
What engineer came up with
this cool protein?

38
00:03:06,600 --> 00:03:08,320
AUDIENCE: MIT engineers.

39
00:03:08,320 --> 00:03:11,600
PROFESSOR: MIT engineers,
yeah.

40
00:03:11,600 --> 00:03:14,270
Not a chance.

41
00:03:14,270 --> 00:03:15,960
Not a chance.

42
00:03:15,960 --> 00:03:19,010
This is a really tough feat.

43
00:03:19,010 --> 00:03:21,610
This is something that can only
be done by the smartest

44
00:03:21,610 --> 00:03:22,940
engineers on the planet.

45
00:03:22,940 --> 00:03:25,550
And MIT engineers are
unfortunately only the

46
00:03:25,550 --> 00:03:27,925
smartest human engineers
on the planet.

47
00:03:32,910 --> 00:03:35,196
Who came up with this
is E. coli.

48
00:03:35,196 --> 00:03:36,940
AUDIENCE: So you found it
somewhere in nature?

49
00:03:36,940 --> 00:03:37,490
PROFESSOR: Sorry?

50
00:03:37,490 --> 00:03:39,020
AUDIENCE: You found it
somewhere in nature?

51
00:03:39,020 --> 00:03:40,950
PROFESSOR: Of course you find
it somewhere in nature.

52
00:03:40,950 --> 00:03:43,860
Almost everything important
that we say molecular

53
00:03:43,860 --> 00:03:47,640
biologists have come up with, it
means molecular sat at the

54
00:03:47,640 --> 00:03:52,120
feet of the true masters,
bacteria, and learned from the

55
00:03:52,120 --> 00:03:54,310
true masters.

56
00:03:54,310 --> 00:03:57,080
This protein is found
in nature.

57
00:03:57,080 --> 00:03:58,330
And it's found in E. coli.

58
00:04:01,810 --> 00:04:07,090
In fact, it's found in E. coli
strain R. And it was the first

59
00:04:07,090 --> 00:04:10,600
such protein found in E.
coli strain R, so it

60
00:04:10,600 --> 00:04:12,290
gets the name EcoR1.

61
00:04:16,269 --> 00:04:19,269
And it cuts the DNA like this.

62
00:04:19,269 --> 00:04:21,140
Pretty cool.

63
00:04:21,140 --> 00:04:23,060
Pretty cool.

64
00:04:23,060 --> 00:04:33,820
Now, it turns out that E.
coli has this EcoR1.

65
00:04:33,820 --> 00:04:35,860
How often does E. coli--

66
00:04:35,860 --> 00:04:38,875
so whenever I take EcoR1, this
protein, purified from E.

67
00:04:38,875 --> 00:04:43,520
coli, and I add it to DNA it
always cuts at this site,

68
00:04:43,520 --> 00:04:46,970
which we call an EcoR1 site.

69
00:04:46,970 --> 00:04:48,760
How frequently do we
expect, what's the

70
00:04:48,760 --> 00:04:51,120
frequency of EcoR1 sites?

71
00:04:54,470 --> 00:04:59,430
G-A-A-T-T-C, how often will
that occur at random?

72
00:05:02,510 --> 00:05:03,243
One in--

73
00:05:03,243 --> 00:05:04,030
AUDIENCE: Two to the sixth?

74
00:05:04,030 --> 00:05:05,520
PROFESSOR: One in two
to the sixth?

75
00:05:05,520 --> 00:05:07,280
How many letters do I have?

76
00:05:07,280 --> 00:05:08,790
AUDIENCE: Four, oh,
four to the sixth.

77
00:05:08,790 --> 00:05:10,090
PROFESSOR: One in four
to the sixth.

78
00:05:10,090 --> 00:05:13,850
My frequency should be about
one in four to the sixth,

79
00:05:13,850 --> 00:05:15,380
which is about what?

80
00:05:15,380 --> 00:05:17,180
What's four to the sixth?

81
00:05:17,180 --> 00:05:18,100
It's two to the 12th.

82
00:05:18,100 --> 00:05:19,981
It's about 4,000.

83
00:05:19,981 --> 00:05:21,830
It's about one in
4,000 letters.

84
00:05:21,830 --> 00:05:25,028
One in 4,000 bases.

85
00:05:25,028 --> 00:05:26,070
So it's very convenient.

86
00:05:26,070 --> 00:05:28,780
One in every 4,000 bases
it'll roughly cut.

87
00:05:28,780 --> 00:05:35,000
It'll cut at roughly
one 4,000 bases.

88
00:05:35,000 --> 00:05:37,290
Why doesn't E. coli
cut its own DNA?

89
00:05:43,370 --> 00:05:45,020
If it's got this protein
floating around in its cell,

90
00:05:45,020 --> 00:05:48,610
why isn't it chopping
up its own DNA?

91
00:05:48,610 --> 00:05:51,370
Doesn't have G-A-A-T-T-C?

92
00:05:51,370 --> 00:05:53,480
Yeah, the problem is
it's so frequent.

93
00:05:53,480 --> 00:05:54,860
That'd be really hard
to make sure--

94
00:05:54,860 --> 00:05:57,680
I mean, E. coli has 4 million
letters in its genome.

95
00:05:57,680 --> 00:05:59,500
This should cut every
4,000 bases.

96
00:05:59,500 --> 00:06:02,430
You expect about 1,000
such sequences.

97
00:06:02,430 --> 00:06:05,450
It might be hard to arrange
not to cut--

98
00:06:05,450 --> 00:06:07,600
not to have any such
sequences.

99
00:06:07,600 --> 00:06:11,125
It's a good idea, is not to have
any, but an alternative--

100
00:06:11,125 --> 00:06:12,610
AUDIENCE: [INAUDIBLE].

101
00:06:12,610 --> 00:06:13,940
PROFESSOR: It protects them.

102
00:06:13,940 --> 00:06:17,830
It turns out E. coli, instead
of avoiding the sequence

103
00:06:17,830 --> 00:06:20,690
altogether, has another
trick up its sleeve.

104
00:06:20,690 --> 00:06:25,330
E. coli protects this sequence
whenever it occurs.

105
00:06:25,330 --> 00:06:29,215
So it turns out that whenever
you have a stretch of the E.

106
00:06:29,215 --> 00:06:38,325
coli genome that has this
G-A-A-T-T-C in it, what E.

107
00:06:38,325 --> 00:06:41,020
coli does is it puts--

108
00:06:41,020 --> 00:06:43,800
I'm just writing M-E here
for a methyl group.

109
00:06:43,800 --> 00:06:45,400
Right, C-H three up there.

110
00:06:45,400 --> 00:06:46,590
It puts a methyl group--

111
00:06:46,590 --> 00:06:48,010
I'll write C-H three.

112
00:06:48,010 --> 00:06:49,510
There we go.

113
00:06:49,510 --> 00:06:54,620
It puts a methyl group on
the A, that middle A.

114
00:06:54,620 --> 00:06:59,900
Well, that is a cute trick that
E. coli uses, putting a

115
00:06:59,900 --> 00:07:01,150
methyl group there.

116
00:07:03,620 --> 00:07:06,630
Because what happens is, when
there's a methyl group, right

117
00:07:06,630 --> 00:07:11,930
at that position, the enzyme
no longer recognizes and no

118
00:07:11,930 --> 00:07:13,180
longer cuts there.

119
00:07:15,970 --> 00:07:18,770
So that's kind of clever.

120
00:07:18,770 --> 00:07:21,555
E. coli makes this protein
that can recognize

121
00:07:21,555 --> 00:07:25,660
G-A-A-T-T-C, but it has a
second protein that puts

122
00:07:25,660 --> 00:07:27,200
methyl groups there.

123
00:07:27,200 --> 00:07:30,830
And this protein happens
by accident

124
00:07:30,830 --> 00:07:32,540
to be called a methylase.

125
00:07:36,720 --> 00:07:38,030
It has a methylase.

126
00:07:38,030 --> 00:07:40,930
And the methylase protects
that sequence.

127
00:07:40,930 --> 00:07:44,180
So now, this is really cool
engineering, but kind of dumb.

128
00:07:44,180 --> 00:07:45,230
What's it doing there?

129
00:07:45,230 --> 00:07:47,230
It has something that cuts the
sequence and it protects the

130
00:07:47,230 --> 00:07:50,550
sequence, why bother
having this?

131
00:07:50,550 --> 00:07:52,392
Yeah?

132
00:07:52,392 --> 00:07:57,232
AUDIENCE: You can use it to cut
it at places to unwrap the

133
00:07:57,232 --> 00:07:58,200
true strands.

134
00:07:58,200 --> 00:07:59,410
PROFESSOR: That's an
interesting idea.

135
00:07:59,410 --> 00:08:02,140
We could use it to cut our DNA
and open it up to unravel our

136
00:08:02,140 --> 00:08:03,010
true strands.

137
00:08:03,010 --> 00:08:03,820
It's a thought.

138
00:08:03,820 --> 00:08:04,710
Yes?

139
00:08:04,710 --> 00:08:08,150
AUDIENCE: To protect the
bacteria from viruses?

140
00:08:08,150 --> 00:08:11,030
PROFESSOR: Protect the bacteria
from viruses.

141
00:08:11,030 --> 00:08:12,485
How do you protect yourself
from viruses?

142
00:08:15,450 --> 00:08:17,540
Well, you have an immune system
with immune cells and

143
00:08:17,540 --> 00:08:18,570
antibodies and all that.

144
00:08:18,570 --> 00:08:20,930
Does E. coli have an
immune system?

145
00:08:20,930 --> 00:08:22,215
Why doesn't it have
immune cells?

146
00:08:24,840 --> 00:08:26,010
Because it's like one cell.

147
00:08:26,010 --> 00:08:28,710
How's it going to have an
immune system, right?

148
00:08:28,710 --> 00:08:30,320
So suppose E. coli
gets a cold.

149
00:08:30,320 --> 00:08:32,700
Suppose it gets infected
by a virus.

150
00:08:32,700 --> 00:08:35,610
How's it going to
protect itself?

151
00:08:35,610 --> 00:08:39,830
Cut at a frequently occurring
DNA sequence.

152
00:08:39,830 --> 00:08:45,720
Now the virus, of course, isn't
methylated there, bingo.

153
00:08:45,720 --> 00:08:47,100
That's how it tells its own--

154
00:08:47,100 --> 00:08:49,080
you can tell cell
from an invader.

155
00:08:52,710 --> 00:08:55,520
E. coli can tell cell from
an invader because it's

156
00:08:55,520 --> 00:08:59,590
methylated its own G-A-A-T-T-C
sites, but the virus isn't

157
00:08:59,590 --> 00:09:01,130
methylated there.

158
00:09:01,130 --> 00:09:02,980
Way cool.

159
00:09:02,980 --> 00:09:04,880
This is an immune system
for E. coli.

160
00:09:08,620 --> 00:09:10,440
Now, it turns out-- so
this is protection.

161
00:09:10,440 --> 00:09:21,460
These restriction enzymes
protect E. coli from viruses.

162
00:09:30,170 --> 00:09:32,750
It turns out that E.
coli is not alone

163
00:09:32,750 --> 00:09:34,580
in this clever trick.

164
00:09:34,580 --> 00:09:38,770
It turns out that other bacteria
have also thought of

165
00:09:38,770 --> 00:09:40,270
this trick.

166
00:09:40,270 --> 00:09:43,870
So it turns out that there is
another restriction enzyme

167
00:09:43,870 --> 00:09:54,040
that cuts at G-G-A-T-C-C. And
on the other strand it goes

168
00:09:54,040 --> 00:10:01,750
G-G-A-T-C-C. It, again, cuts in
that distinctive pattern.

169
00:10:01,750 --> 00:10:04,005
And it's called BamH1.

170
00:10:08,971 --> 00:10:10,650
And there's another guy.

171
00:10:10,650 --> 00:10:20,060
And he cuts at A-A-G-C-T-T,
A-A-G-C-T-T. And it

172
00:10:20,060 --> 00:10:22,040
also cuts like that.

173
00:10:22,040 --> 00:10:24,820
And it's called HindIII.

174
00:10:28,004 --> 00:10:32,960
And there's some that
cut at G-A-T-C, just

175
00:10:32,960 --> 00:10:34,390
the four letter word.

176
00:10:36,950 --> 00:10:39,170
And they cut like that.

177
00:10:39,170 --> 00:10:54,790
And there's some that cut at
C-A-G-C-T-G, C-A-G-C-T-G, and

178
00:10:54,790 --> 00:10:58,380
this, cuts smack
in the middle.

179
00:10:58,380 --> 00:11:02,690
In other words, there's a wide
number of different tricks.

180
00:11:02,690 --> 00:11:04,370
Some cut at six bases.

181
00:11:04,370 --> 00:11:05,740
Some cut at four bases.

182
00:11:05,740 --> 00:11:07,590
Some cut at eight bases.

183
00:11:07,590 --> 00:11:09,710
Some cut leaving an overhang.

184
00:11:09,710 --> 00:11:11,640
Some cut smack in the middle.

185
00:11:11,640 --> 00:11:15,670
Some cut leaving the overhang
in the other direction.

186
00:11:15,670 --> 00:11:18,240
Some allow a degenerate
base in the middle.

187
00:11:18,240 --> 00:11:21,350
It doesn't care which base
is in the middle.

188
00:11:21,350 --> 00:11:24,110
There's a zillion different
solutions that bacteria have

189
00:11:24,110 --> 00:11:26,390
come up with for their
immune system.

190
00:11:26,390 --> 00:11:30,390
And so, if I want to cut up some
human DNA all I need is

191
00:11:30,390 --> 00:11:35,420
say, this protein EcoR1 or BamH1
or HindIII or MVL 1 or

192
00:11:35,420 --> 00:11:37,695
PVU 2 or et cetera.

193
00:11:37,695 --> 00:11:40,710
And I can do that by growing
up E. coli and

194
00:11:40,710 --> 00:11:41,960
purifying the protein.

195
00:11:44,810 --> 00:11:48,010
And if I wanted HindIII, I
would grow up haemophilus

196
00:11:48,010 --> 00:11:51,060
influenza and purify
the protein.

197
00:11:51,060 --> 00:11:53,890
So in a molecular biology lab,
today, if you want to cut up

198
00:11:53,890 --> 00:11:58,000
human DNA, you could grow up
some E. coli and purify EcoR1

199
00:11:58,000 --> 00:11:59,250
or haemophilus influenza.

200
00:12:01,260 --> 00:12:04,710
And that is indeed what ancient
molecular biologists

201
00:12:04,710 --> 00:12:09,360
did in prehistoric days in
the 1970s and 1980s.

202
00:12:09,360 --> 00:12:13,000
They would purify their own
restriction enzymes.

203
00:12:13,000 --> 00:12:14,090
They're still alive today.

204
00:12:14,090 --> 00:12:15,400
You can talk to them.

205
00:12:15,400 --> 00:12:17,140
There are many of them
on the faculty.

206
00:12:17,140 --> 00:12:20,010
And they'll tell you how it put
hair on their chest to be

207
00:12:20,010 --> 00:12:22,265
able to purify their own
restriction enzymes.

208
00:12:27,610 --> 00:12:30,150
What do you do today?

209
00:12:30,150 --> 00:12:33,275
Order it online from
the catalog, right?

210
00:12:33,275 --> 00:12:36,065
You know, there's the
catalog, the New

211
00:12:36,065 --> 00:12:38,480
England Bio Labs catalog.

212
00:12:38,480 --> 00:12:39,730
Let's see what we got here.

213
00:12:43,800 --> 00:12:53,470
Restriction enzymes, modifying
enzymes, polymerases, all

214
00:12:53,470 --> 00:12:59,150
right, EcoR1, sale on
EcoR1 right now.

215
00:12:59,150 --> 00:13:04,070
$100 buys you 10,000
units of EcoR1.

216
00:13:04,070 --> 00:13:05,030
It's in the catalog.

217
00:13:05,030 --> 00:13:05,780
You can go online.

218
00:13:05,780 --> 00:13:06,270
You can order it.

219
00:13:06,270 --> 00:13:08,390
You can have it tomorrow
by FedEx.

220
00:13:08,390 --> 00:13:10,480
So, but that's how it works.

221
00:13:10,480 --> 00:13:11,810
It's in the catalog.

222
00:13:11,810 --> 00:13:15,930
So you can get any restriction
enzyme you want to cut DNA

223
00:13:15,930 --> 00:13:17,180
anywhere you want to.