Awards in the scientific disciplines of physics and chemistry require that the significance of achievements being recognized is &quot;tested by time.&quot; In practice it means that the lag between the discovery and the award is typically on the order of 20 years and can be much longer. she is the only woman to receive an unshared Nobel Prize in that category
13-2 manipulating DNA
Chapter 13 -2Manipulating DNA P 322
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
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 Power source Longer fragments Shorter fragmentsMixture of DNA Porous gelfragments - DNA is placed at one end of a porous gel ???? Agarose gel
3. Gel electrophoresis (separate DNA) DNA plus restriction enzyme Power source Longer fragments Shorter fragmentsMixture of DNA Porous gelfragments - A current is applied to the gel
3. Gel electrophoresis (separate DNA) DNA plus restriction enzyme Power source Longer fragments Shorter fragmentsMixture of DNA Porous gelfragments - DNA molecules are negatively charged and move toward positive end of gel
3. Gel electrophoresis (separate DNA) DNA plus restriction enzyme Power source Longer fragments Shorter fragmentsMixture of DNA Porous gelfragments - 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: 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.
• 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
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.
Each year, hundreds ofimmigrants lose their liveswhile slipping across theborder into Arizona’s SonoranDesert. DNA forensics ispiecing together the identitiesand 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 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。
Interesting PCR songThere 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 its 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.
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℃
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
steps1) 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’
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