Chapter 13 -2Manipulating DNA

P 322

Genetic engineering Genes are isolated, modified, and

inserted into an organism Basic techniques:

1. Extracting DNA from cells2. Cutting DNA into smaller pieces3. Gel electrophoresis (separate DNA)4. PCR (amplifying DNA)5. Making recombinant DNA

Basic techniques of genetic engineering

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

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

3. Gel electrophoresis (separate DNA)

DNA plus restriction enzyme

Mixture of DNA fragments

Porous gel

Power source

Longer fragments

Shorter fragments

- DNA is placed at one end of a porous gel

Agarose gel????

3. Gel electrophoresis (separate DNA)

DNA plus restriction enzyme

Mixture of DNA fragments

Porous gel

Power source

Longer fragments

Shorter fragments

- A current is applied to the gel

3. Gel electrophoresis (separate DNA)

DNA plus restriction enzyme

Mixture of DNA fragments

Porous gel

Power source

Longer fragments

Shorter fragments

- DNA molecules are negatively charged and move toward positive end of gel

3. Gel electrophoresis (separate DNA)

DNA plus restriction enzyme

Mixture of DNA fragments

Porous gel

Power source

Longer fragments

Shorter fragments

- Smaller molecules move faster than larger ones

Application: DNA Fingerprints

Unique array of DNA fragments Steps:

Restriction enzymes to cut Run gel electrophoresis

Application: Identify criminal suspects

Identify bodies

Determine paternity

DNA fingerprinting is a technique that compares DNA from different sources to identify individuals and show their relationship.

More similarities the DNA shares, more close the relationship is.

• DNA fingerprinting: DNA 指纹图谱• 就像每个人都有不同的指纹一样，每个人的 DNA 图谱都不一样，通过比较

DNA图谱之间的相似度和差异度，可以确定人与人之间的血缘关系

Genetic marker is the fingerprint of DNA, which could be passed to offspring.

Genetic marker is more likely to be an exact match between relatives than between unrelated individuals.

• Genetic marker: 遗传标记

• 遗传标记广泛存在于 DNA上，可以遗传给下一代

• 亲缘关系越近，完全一样的遗传标记数量就越多，反之则越少

• 如果把不同的人比作不同的城市，那么遗传标记就是这座城市的地标

Forensic Investigation Genetic markers are

separated to display different patterns of bands.

In this example, DNA from suspect 2 matches DNA found at the crime scene, but DNA suspect 1 does not match.

• 遗传标记被广泛应用于Forensic Investigation (法医鉴定）中

• 多个嫌疑人的 DNA被提取出来，通过与犯罪现场得到的 DNA的条带对比可以确定曾出现在犯罪现场的嫌疑人

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.

• 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

Paternity profiling DNA fingerprinting to determine

paternity uses the concept that each band (genetic marker) of a child must correspond with a band (genetic marker) of the father or the mother.

Paternity profiling: 亲子鉴定亲子鉴定是 DNA指纹的一项重要应用。

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。

Check your knowledge

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)

PCR song

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."

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.

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.

Polymerase Chain Reaction (PCR) Reagents: Templates, buffer, primers, nucleotides(dNTP), DNA

polymerases

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

Polymerase Chain Reaction (PCR) Applications: Detect mutations ， Recombine ， Paternity profiling and

Forensic Investigation

Double-stranded DNA to copy

DNA heated to 90°– 94°C, make DNA unwind

Primers added

Mixture cooled; base-pairing of primers and ends of DNA strands

DNA polymerasesassemble new DNA strands at 72℃

1. Denaturing: Heating to unwind DNA

2. Annealing: Cooling to bind primers to ends of single strands

3. Extending: DNA

polymerase (Taq) uses free nucleotides to create complementary strands

Steps

5. Making recombinant DNA( DNA 重组 )

Cut and recombine DNA from different species and insert it into bacteria, yeast, plants or other mammalian cells.

The cells copied the inserted DNA as their own DNA rapidly

steps1) Restriction enzyme to cut

5’

3’

G

C T T A A

A A T T C

G

G A A T T C

C T T A A G3’

5’

one DNA fragment another DNA fragment

3’

5’

2) DNA ligase to ligate

5’

3’

3’

5’

G A A T T C

C T T A A G

G A A T T C

C T T A A G

DNA ligase action

3) Insert recombinant DNA into cells