ผศ. ดร. พญ. วรพรรณ ศ ร ว ฒนอ กษร (พ ล กโป ง)

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1 ผศ. ดร. พญ. วรพรรณ ศ ร ว ฒนอ กษร (พ ล กโป ง)

2 Molecular biology techniques DNA extraction Electrophoresis Southern blotting DNA fingerprinting Polymerase chain reaction (PCR) DNA sequencing DNA cloning

3 DNA extraction Genomic DNA can be extracted from any nucleated tissue and white blood cell. 1. Break down the cell walls. 2. Break down the lipid molecules of the cell and nuclear membranes, releasing the contents of the cell, including the DNA. 3. Precipitate DNA in alcohol.

4 DNA Extraction Disruption of cell membrane & nuclear membrane using sodium dodecyl sulfate (SDS) & proteinase K Release DNA & other cellular components

5 DNA Extraction Disruption of the cell membrane sodium dodecyl sulfate (SDS) When SDS comes close to the cell, it captures the lipids and proteins.

6 DNA Extraction Disruption of the cell membrane proteinase K The DNA in the nucleus of the cell is molded, folded, and protected by proteins. Proteinase K cuts the proteins away from the DNA.

7 - In water, DNA is soluble. - Alcohol is less dense than water, so it floats on top. - The protein and grease parts stay in the bottom, watery layer. DNA - DNA prefers the top, alcohol layer. protein - DNA is a long, stringy molecule and that likes to clump together. RNA

8 DNA Extraction DNA negative charge more soluble & stable in salt solution Medium a buffered saline solution containing EDTA (Ethylenediaminetetra acetic acid) EDTA 1. binds Cd 2+, Mg 2+, Mn 2+ that could form salt with the anionic phosphate groups of the DNA 2. inhibits deoxyribonucleases

9 Restriction enzymes DNA-cutting enzymes found in bacteria often called restriction endonucleases recognize & cut DNA only at a particular sequence of nucleotides (4, 6 or 8 bp) : restriction ti site part of mechanism for bacteria to recognise foreign DNA Palindromes - the sequences recognized by restriction enzymes & have symmetry y of the form 5 GAATTC 3 3 CTTAAG 5 or AGCT TCGA or GGCC CCGG

10 Restriction enzymes Restriction endonuclease

11 Restriction enzymes Restriction i endonuclease sticky ends

12 Restriction enzymes Restriction endonuclease cut endonuclease CCCGGG CCC GGGCCC GGG GGG CCC blunt ends

13 Restriction enzymes Restriction enzymes cut DNA ONLY if the exact Restriction enzymes cut DNA ONLY if the exact recognition sequence is present :

14 Restriction enzymes Examples source enzyme recognition fragment site end E.Coli RY13 EcoRI G/AATTC sticky Bacillus amyloliquifaciens H BamHI G/GATCC sticky Serracia marcescens Sb SmaI CCC/GGG blunt Providentia stuartii PstI CTGCA/G sticky

15 Gel Electrophoresis electro = flow of electricity phoresis (Greek) = to carry across gel : colloid : suspension of tiny particles in a medium, occurring in a solid form, like gelatin gel electrophoresis : the separation of charged particles located in a gel when an electric current is applied charged particles : DNA, amino acids, peptides, etc.

16 Gel Electrophoresis Why do gel electrophoresis? DNA cut by restriction enzymes a mixture of DNA fragments (different lengths) It is useful to be able to separate the DNA pieces for recovering particular pieces of DNA for forensic work or for sequencing

17 Gel Electrophoresis agarose Gel polysaccharide made from seaweed acrylamide water soluble, cross-linked polymer sharper bands

18 Gel Electrophoresis How does it work? DNA : negatively charged (remember, DNA for Negative) In an electrical field, DNA migrates toward the positive electrode Smaller pieces of DNA can travel faster than larger pieces

19 Gel Electrophoresis "Mice" run faster through the forest than "elephants"

20 Gel Electrophoresis "Mice" run faster through the forest than "elephants"

21 Gel Electrophoresis "Mice" run faster through the forest than "elephants"

22 Gel Electrophoresis Negatively el charged nucleic acids Place mixture on an agarose or polyacrylamide l gel Apply electric field + Molecules move through pores in gel at a rate inversely proportional to their chain length + - gel particle - pores

23 Gel Electrophoresis A gel being run positive electrode comb agarose block DNA loaded in wells in the agarose black background buffer

24 Gel Electrophoresis A gel as seen under UV light

25 Gel Electrophoresis Fragments of DNA seperated by gel electrophoresis

26 Gel Electrophoresis DNA ladder : DNA fragments of known size used for comparison The DNA band of interest can be cut out of the gel and the DNA can be extracted. t Or DNA can be removed from the gel by Or DNA can be removed from the gel by Southern Blotting.

27 Detection of nucleic acid & proteins Nucleic acid hybridisation Southern blotting Northern blotting Antibodies as probes for proteins Western blotting

28 Nucleic acid hybridisation Hybridisation : the process of joining two complementary strands Hybridisation probe : a single stranded piece of radioactive DNA (or RNA) complementary to part of the DNA fragment of interest (target DNA) When added to the target DNA, the probe will hydrogen bond to it

29 Nucleic acid hybridisation DNA is denatured by heating Renaturation on cooling (anneal) probe radioactive hybridisation

30 Nucleic acid hybridisation DNA is denatured by heating to 95 C single strand molecules l radiolabeled probe is added, 65 C complementary DNA strands renature or hybridise radiolabeled probe can be detected as radioactive double-stranded molecules

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32 Southern blotting Gel of restriction enzyme digested human DNA

33 Southern blotting Hybridisation with probe

34 Southern blotting Resulting autoradiogram

35 Southern blotting Is suspect #1 guilty?

36 Southern blotting DNA fingerprinting g The entire sequence of DNA in each human : never be identical except identical twins So each person s DNA is as unique as a fingerprinti

37 DNA fingerprinting g

38 DNA fingerprinting identify rapists and other criminals determine paternity; that is, who the father of the child really is immigration control

39 Polymerase chain reaction (PCR) selectively and repeatedly replicating defined DNA from a DNA mixture amplifies DNA segment exponentially requires knowledge of sequences desired size of product determined by choice of primer positions 2 primers designed (15-25 nucleotides long)

40 PCR

41 PCR

42 PCR Exponential amplification Number of products = 2 n Where n is the number of cycles cycles # products ,048, x x

43 PCR Steps in PCR denaturation (melting) (94 C) annealing of primers (55 C) polymerase extension (72 C) (polymerisation) Repeats Cycles!

44 PCR Melting

45 PCR Anneal primers primers : short (18-24 nt), synthetic ti oligonucleotides l sequence : complementary to target strands distance between 5 ends determines size of PCR products

46 PCR Polymerase extension

47 PCR Polymerase extension

48 PCR Products from one strand parental DNA strand = template for new strand (beginning at 5 end of primer) melt and anneal new primers

49 PCR Products of second PCR cycle parental strand (A) remains first product strand (B) copied from parental second product strand (C) copied from first product (B)

50 PCR Amplification Parental strand (A) never completely copied into long strand. First product (B) has defined 5 end. Second product (C) has both defined 5 and ddefined d3 ends. After several cycles of PCR, almost all After several cycles of PCR, almost all products are like C.

51 PCR One tube of PCR reaction contains : DNA template t a pair of oligonucleotide primers DNA polymerase DNA DNA precursors (4 deoxynucleotides : datp, dctp, dgtp & dttp)

52 PCR Taq DNA polymerase prepared from Thermus aquaticus, a thermophilic bacteria Half-life life min. at 94 C optimal polymerase activity at 72 C PCR d t il i li d b d f PCR products easily visualised as a band of a specific size by agarose gel electrophoresis.

53 2 Strategies DNA sequencing Maxam-Gilbert label one end of DNA chemically break DNA at a specific base separate products by gel electrophoresis Sanger (dideoxy) use polymerase & primer to synthesise DNA stop at one base (dideoxy) separate products by gel electrophoresis

54 DNA sequencing Why dideoxy? Polymerase requires template base-paired primer with 3 OH deoxynucleotides (dntps) Dideoxy (ddntp) works as dntp substrate After incorporation, primer lacks 3 OH Therefore, ddntp terminates chain

55 DNA sequencing Dideoxynucleotides

56 DNA sequencing

57 DNA sequencing

58 DNA sequencing Overview of Sanger Sequencing

59 DNA sequencing Automated Sequencing Use fluorescently labeled ddntps. Each base uses a different colour. One reaction instead of four. Load onto gel inside a scanning fluorimeter. Read nt per DNA. One person can process up to 40 DNAs per day. Expand capacity

60 DNA sequencing Automated sequencing output

61 Recombinant DNA Combine plasmid vector with any DNA fragments Use sticky ends or blunt ends produced by restriction enzymes as sites for recombination Use DNA ligase to attach two DNA molecules ligation

62 Recombinant DNA using a restriction enzyme that cuts the double strand at a particular point the same enzyme is used to cut a second piece of DNA mix the fragments together the complementary ends of each strand will bind with those of the other, forming a recombinant DNA molecule

63 Vectors Cloning vector Plasmid Bacteriophage vectors Cosmid vectors Bacteriophage P1 PAC (P1 artificial chr) BAC (bacterial artificial chr) YAC (yeast artificial chr) size of insert 0-10 kb 0-23 kb kb kb kb up to 300 kb Mb

64 Plasmid (eg. pbr 322, puc 19,18,8,9) small circular double-stranded DNA contain very few genes intracellular vertically distributed to daughter cells following host cell divisioni i horizontally transferred to neighboring cells during bacterial conjugation an ori sequence a dominant selectable marker (antibiotic resistance) unique restriction enzyme cleavage site

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66 The cell-based DNA cloning involves 4 steps 1. Construction ti of recombinant DNA molecules l by ligation of desired DNA fragments (target DNA) to a vector cutting the target DNA & replicon molecules with specific restriction endonucleases j i i h diff DNAf i joining the different DNA fragments using the enzyme DNA ligase

67 DNA cloning Construction ti of recombinant DNA molecules l

68 DNA cloning 2. Transformation Recombinant DNA molecules are transferred into host cells (often bacterial or yeast cells). The vector can undergo DNA replication independently of the host cell chromosome(s). Host cells : competent t cells (E.coli) CaCl 2 2, MgCl 2, Rhubidium chloride

69 DNA cloning 3. Grow clones The transformed cells are plated out by spreading on an agar surface. Clones A clone is a group of genetically identical individuals. id 4. Identification of positive clones phenotypic expression DNA hybridisation immunology

70 DNA cloning

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