1. Chapter 13 -2
Manipulating DNA
P 322

2. Genetic engineering
 Genes are isolated, modified, and
inserted into an organism
 Basic techniques:
1. Extracting DNA from cells
2. Cutting DNA into smaller pieces
3. Gel electrophoresis (separate DNA)
4. PCR (amplifying DNA)
5. Making recombinant DNA

3. Basic techniques of genetic engineering

4. 1. Extracting DNA from cells
• Break open cells to access DNA
Mechanical methods: to grind cells, sonication, etc
Chemical methods: Cell Lysis Buffer, detergent, etc
• Purify DNA from cell debris
Precipitate DNA
Extract DNA binding proteins

5. 2. Cutting DNA into smaller pieces
Restriction enzyme: cutting DNA at a specific
sequence; forming sticky ends
Restriction enzyme
EcoR I cuts the DNA
into fragments.
Specific sequence for
EcoRI : G AATTC
Read in 5’ 3’
Sticky ends

6. 3. Gel electrophoresis (separate DNA)
DNA plus restriction
enzyme Power
source
Longer
fragments
Shorter
fragments
Mixture of DNA Porous gel
fragments
- DNA is placed at one end of a porous gel
???? Agarose gel

7. 3. Gel electrophoresis (separate DNA)
DNA plus restriction
enzyme Power
source
Longer
fragments
Shorter
fragments
Mixture of DNA Porous gel
fragments
- A current is applied to the gel

8. 3. Gel electrophoresis (separate DNA)
DNA plus restriction
enzyme Power
source
Longer
fragments
Shorter
fragments
Mixture of DNA Porous gel
fragments
- DNA molecules are negatively charged
and move toward positive end of gel

9. 3. Gel electrophoresis (separate DNA)
DNA plus restriction
enzyme Power
source
Longer
fragments
Shorter
fragments
Mixture of DNA Porous gel
fragments
- Smaller molecules move faster than larger ones

13. Application: DNA Fingerprints
 Unique array of DNA fragments
 Steps:
 Restriction enzymes to cut
 Run gel electrophoresis
 Application:
 Identify criminal suspects
 Identify bodies
 Determine paternity

14. • DNA fingerprinting: DNA  DNA fingerprinting is a
指纹图谱 technique that compares
• 就像每个人都有不同的指 DNA from different sources
纹一样，每个人的 DNA 图 to identify individuals and
谱都不一样，通过比较 show their relationship.
DNA 图谱之间的相似度和  More similarities the DNA
差异度，可以确定人与人 shares, more close the
之间的血缘关系 relationship is.

15. • Genetic marker: 遗传标  Genetic marker is the
记 fingerprint of DNA, which
• 遗传标记广泛存在于 DNA could be passed to offspring.
• 上，可以遗传给下一代
如果把不同的人比作不同  Genetic marker is more likely
的城市，那么遗传标记就 to be an exact match between
是这座城市的地标 relatives than between
• 亲缘关系越近，完全一样 unrelated individuals.
的遗传标记数量就越多，
反之则越少

16. Forensic Investigation
• 遗传标记被广泛应用于  Genetic markers are
Forensic Investigation ( 法 separated to display
医鉴定）中 different patterns of bands.
• 多个嫌疑人的 DNA 被
提取出来，通过与犯罪  In this example, DNA from
现场得到的 DNA 的条 suspect 2 matches DNA
带对比可以确定曾出现 found at the crime scene,
在犯罪现场的嫌疑人 but DNA suspect 1 does
not match.

17. Each year, hundreds of
immigrants lose their lives
while slipping across the
border into Arizona’s Sonoran
Desert. DNA forensics is
piecing together the identities
and stories of the dead.

18. • DNA 指纹图谱也被用于鉴定
无法识别的尸体
• 通过尸体 DNA 采样与亲属
DNA 采样的指纹图谱对比可
以确定死者身份

19. Paternity profiling
 DNA fingerprinting to determine Paternity profiling: 亲子
paternity uses the concept that each 鉴定
band (genetic marker) of a child must
correspond with a band (genetic 亲子鉴定是 DNA 指纹
marker) of the father or the mother. 的一项重要应用。
亲子鉴定的原理是父母
的 DNA 指纹条带不一
定会遗传给孩子，但是
孩子的 DNA 指纹条带
一定可以从父母的
DNA 指纹图谱发现相
应的条带。

20. Check your knowledge

21. 4. Polymerase Chain Reaction
( 聚合酶链式反应 )
 A fast way to amplify
DNA
 In test tubes, not in
the living cells (in vitro)

22. PCR song

23. Interesting PCR song
There was a time when to amplify DNA,
You had to grow tons and tons of tiny cells.
Then along came a guy named Dr. Kary Mullis,
Said you can amplify in vitro just as well.
Just mix your template with a buffer and some primers,
Nucleotides and polymerases, too.
Denaturing, annealing, and extending.
Well it's amazing what heating and cooling and heating will do.
PCR, when you need to detect mutations.
PCR, when you need to recombine.
PCR, when you need to find out who the daddy is.
PCR, when you need to solve a crime."

24. Polymerase Chain Reaction (PCR)
 Once Upon a time:
 When researchers want to study a particular sequence of DNA,
they need many (identical) copies of it. The traditional method
of DNA amplification (using plasmids in vivo) takes a lot of time
to grow cells.

25. Polymerase Chain Reaction (PCR)
 History of PCR:
 In 1983, Dr. Kary Mullis developed PCR which could amplify
DNA in vitro.
• in vivo (Latin for "within the
living") is experimentation using a
whole, living organism.
• in vitro (Latin: " within the glass")
is performed not in a living
organism but in a controlled
environment, such as in a test tube
or Petri dish.

26. Polymerase Chain Reaction (PCR)
 Reagents:
 Templates, buffer, primers, nucleotides(dNTP), DNA
polymerases

27. Polymerase Chain Reaction (PCR)
 Steps:
 95 ºC Denaturing
 50~60 ºC Annealing
 72 ºC Extending

28. Polymerase Chain Reaction (PCR)
 Applications:
 Detect mutations ， Recombine ， Paternity profiling and
Forensic Investigation

29. Double-stranded
DNA to copy
Steps
DNA heated to
• Denaturing: Heating 90°– 94°C, make
DNA unwind
to unwind DNA
Primers added
• Annealing: Cooling to
bind primers to ends of
single strands Mixture cooled;
base-pairing of
• Extending: DNA primers and ends
of DNA strands
polymerase (Taq) uses
free nucleotides to create DNA polymerases
complementary strands assemble new
DNA strands at 72℃

31. 5. Making recombinant DNA( DNA 重组 )
 Cutand recombine DNA from different
species and insert it into bacteria, yeast,
plants or other mammalian cells.
 Thecells copied the inserted DNA as their
own DNA rapidly

32. steps
1) Restriction enzyme to cut
5’ G A A T T C
3’ C T T A A G
one DNA fragment another DNA fragment
5’ G A A T T C 3’
3’ C T T A A G 5’

34. 2) DNA ligase to ligate
5’ G A A T T C 3’
3’ C T T A A G 5’
DNA ligase action
G A A T T C
C T T A AG

36. 3) Insert recombinant DNA into cells