Chapter 6 - Molecular Genetic Techniques

Transcription

1 Chapter 6 - Molecular Genetic Techniques

2 Two objects of molecular & genetic technologies For analysis For generation

3 Molecular genetic technologies! For analysis DNA gel electrophoresis Southern blotting DNA sequencing Automation Northern blotting - in situ hybridization cdna library Microarray Next generation sequencing Beyond the in vitro works

4 Molecular genetic technologies! For manipulation Cloning Next semester! Subcloning (for amplification, mutation, fusion and so on) Enzyme cutting, Ligation, PCR, Cell culture, DNA purification, Gene transformation, Gene transfection (ex. to Cell line) Transgenic animals Viral infected animals Gene editing with CRISPR/CAS9

5 DNA works

6 DNA gel electrophoresis Only by size

7 Southern blotting A technique for DNA detection developed earlier by Edwin Southern Question: Is sequence A there? Which sequence is it? Concept of PROBE! DNA is complementary!

8 Genomic DNA Too long to analyze

9 Restriction enzyme Restriction enzymes, also known as restriction endonucleases, are enzymes that cut a DNA molecule at a particular place. Sticky ends Blunt ends

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11 Southern blotting A technique for DNA detection developed earlier by Edwin Southern

12 Southern blotting

13 DNA sequencing

14 DNA sequencing Frederick Sanger, a pioneer of sequencing. Sanger is one of the few scientists who was awarded two Nobel prizes, one for the sequencing of proteins, and the other for the sequencing of DNA.

15 Sanger method DNA sequencing Think the DNA structure You know the details then how to get the sequence information? Hint: using replication process

16 Sanger method DNA sequencing Think the DNA structure You know the details then how to get the sequence information? Hint: using replication process

17 Sanger method DNA sequencing Think the DNA structure You know the details then how to get the sequence information? Hint: using replication process How to stop the reaction?

18 Sanger method DNA sequencing ddntp (ddatp, ddgtp, ddctp, DDTTP) + radioisotope

19 Sanger method DNA sequencing Fluorescent dye ddntp (ddatp, ddgtp, ddctp, DDTTP) + something?

20 Sanger method DNA sequencing ddntp (ddatp, ddgtp, ddctp, DDTTP) + FL dyes Automation!!

21 Genome sequencing Genomic library

22 RNA works

23 Northern blotting The northern blot is a technique used in molecular biology research to study gene expression by detection of RNA (or isolated mrna) in a sample.

24 Northern blotting RNA isolation RNA quality check RNA gel electrophoresis Membrane transfer Detect a RNA sequence by probe

25 Northern blotting

26 Northern blotting to in situ hybridization If you want to see the RNA expression in tissue itself Hint: immunohistochemistry Instead of the membrane you can use the tissue itself!

27 Northern blotting to in situ hybridization

28 Northern blotting to in situ hybridization

29 Northern blotting to in situ hybridization In situ hybridization in cultured cells (Kim HF, 2006) In situ hybridization in neuronal ganglion (Jang, Kim & Kaang, 2016)

30 cdna library

31 Question Which mrnas are expressed in a specific type of cell? 1. Cell isolation 2. Extract the mrna 3. Then?

32 cdna library What is the important step here? How to make the RNA to DNA? Which kind of primer?

33 A cdna library contains representative copies of cellular mrna sequences. Complementary DNAs (cdnas) are synthesized from all the mrnas expressed in a cell type and cloned into plasmid vectors to generate a cdna library. Steps 1 and 2: Retrovirus reverse transcriptase synthesizes a strand of DNA complementary to each mrna molecule (cdna) off an oligo-dt primer base paired to the mrna polya tail. Steps 3 5: The cdna-mrna hybrid molecules are converted into double-stranded cdna molecules with an oligo-dc oligo-dg double-stranded region at one end and an oligo-dt-oligo-da doublestranded region at the other end. Step 6: Methylation of the cdna protects it from subsequent restriction enzyme cleavage. Step 7: Short double-stranded linker DNA molecules containing a specific restriction enzyme recognition site are ligated to both ends of the cdnas using bacteriophage T4 DNA ligase. Step 8a: Restriction enzyme digestion specific for the attached linker generates cdna molecules with sticky ends. Step 8b: Plasmid DNA is treated with the same restriction enzyme to produce appropriate polylinker sticky ends. Step 9: Cut plasmid vectors and the collection of cdnas are mixed at a 1:1 ratio and joined covalently by DNA ligase. The recombinant DNA plasmids are transformed into E. coli cells for propagation.

34 If you want to amplify it You can use PCR also!

35 The polymerase chain reaction (PCR) is widely used to amplify DNA regions with known flanking sequences. What re necessary? Primer DNA polymerase dntp Buffer

36 PCR

37 DNA can be amplified by PCR for use in cloning PCR: Primer sequences that are unique to target flanking regions are synthesized to include restriction enzyme recognition sequences not in the target sequence: Primer 1 contains a BamHI recognition sequence Primer 2 contains a HindIII recognition sequence PCR for 20 cycles [Note: only one of the two strands is shown for simplicity.] Cloning: PCR-amplified sequences are cut with BamHI and HindIII, generating sticky ends. Fragments are ligated into polylinker region of a similarly cut plasmid. Recombinant vector is transformed into E. coli.

38 What do you want to do with cdna library? Cell #1 Cell #2 Make questions Which genes are expressed differentially?

39 Using DNA chip

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41 NEXT GENERATION SEQUENCING

42 Generation of clusters of identical DNA fragments attached to a solid support. Billions of different DNA fragments can be sequenced simultaneously by methods based on PCR. Ligate each end of DNA fragments to be sequenced to doublestranded linkers. Anneal to matching primers that are covalently attached to a solid substrate. (Reactions are optimized to produce as many as discrete, non-overlapping clusters for sequencing.) PCR amplify for 10 cycles to yield ~1000 identical copies of each DNA fragment localized in a small cluster and attached at both ends to the solid substrate.

43 Using fluorescent-tagged deoxyribonucleotide triphosphates for sequence determination. Circled colored dots: the color change reveals which nucleotide was added to the DNA fragment in each reaction cycle.

44 NGS

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46 Molecular genetic technologies! For manipulation Cloning Next semester! Subcloning (for amplification, mutation, fusion and so on) Enzyme cutting, Ligation, PCR, Cell culture, DNA purification, Gene transformation, Gene transfection (ex. to Cell line) Transgenic animals Viral infected animals Gene editing with CRISPR/CAS9

47 Genetically modified animals

48 Read this paper and summarize next generation sequencing method APPLICATIONS OF NEXT-GENERATION SEQUENCING Sequencing technologies the next generation Michael L. Metzker* Abstract Demand has never been greater for revolutionary technologies that deliver fast, inexpensive and accurate genome information. This challenge has catalysed the development of next-generation sequencing (NGS) technologies. The inexpensive production of large volumes of sequence data is the primary advantage over conventional methods. Here, I present a technical review of template preparation, sequencing and imaging, genome alignment and assembly approaches, and recent advances in current and near-term commercially available NGS instruments. I also outline the broad range of applications for NGS technologies, in addition to providing guidelines for platform selection to address biological questions of interest. NATURE REVIEWS GENETICS VOLUME 11 JANUARY Summarize the figure 1 2. Summarize the figure 2 with box 1 3. Summarize the figure 4 with box 1

49 참고 (etc)

50 In Next semester For manipulation Next semester! Subcloning (for amplification, mutation, fusion and so on) Enzyme cutting, Ligation, PCR, Cell culture, DNA purification, Gene transformation,

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54 Ligation of restriction fragments with complementary sticky ends.

55 Basic components of a plasmid cloning vector that can replicate within an E. coli cell.

56 DNA cloning in a plasmid vector permits amplification of a DNA fragment.

57 A yeast genomic library can be constructed in a plasmid shuttle vector that can replicate in yeast and in E. coli

58 6-16 Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of a mutant yeast gene.