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Basic techniques

Basic techniques. --- Nucleic acid hybridization complementary strands will associate and form double stranded molecules --- DNA sequencing Determining the array of nucleotides in a DNA molecule --- Restriction Enzymes These enzymes recognize and cleave DNA at specific sequences

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Basic techniques

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  1. Basic techniques --- Nucleic acid hybridization complementary strands will associate and form double stranded molecules --- DNA sequencing Determining the array of nucleotides in a DNA molecule --- Restriction Enzymes These enzymes recognize and cleave DNA at specific sequences --- Blotting Allows analysis of a single sequence in a mixture of nucleic acids from a single individual --- DNA cloning This allows the isolation and generation of a large number of copies of a given DNA sequence from a single individual --- Transformation Stably integrating a piece of DNA into the genome of an organism --- PCR amplification (making many copies) of a known sequence --- Genetic engineering Altering the DNA sequence of a given piece of DNA --- Genomics Analyzing the entire genome OF INDIVIDUALS

  2. Nucleic acid hybridization Complementary strands of DNA or RNA will specifically associate DNA is heated to 100C, the hydrogen bonds linking the two strands are broken The double helix dissociates into single strands. As the solution is allowed to cool, strands with complementary sequences readily re-form double helixes. This is called Nucleic acid hybridization. 5’ AAAAAATTTTAAAAA 3’ Will associate with 3’ TTTTTTAAAATTTTT 5’ This occurs with complementary DNA/DNA, DNA/RNA, RNA/RNA 5’ AAAAAATTTTAAAAA 3’ Will NOT associate with 3’ TTTTTTAAGATTTTT 5’

  3. Sequencing Genomic DNA Fragment DNA (clone) Sequence fragments Align fragments Build consensus sequence ACGCGATTCA ACGCGATTCA GCGATTCAGGTTA GATTCAGGTTA CAGGTTACCACGC ACGCGTAGCGC TAGCGCA TAGCGCATTACAC ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC

  4. Sequencing Reference Genome- Number of donor DNAs are sequenced Pieces of DNA are sequenced many times Computers are used to overlap the pieces to generate contigs Consensus sequence is reference genome Sequences of individuals will vary from the reference genome ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC Reference Genome ACGCGATTCAGGTTACCACGCGTAGCGCATTACAC SHELBY ACGCGGTTCAGGTTACCACGCGTAGCGCATTACAC JAY ACGCGATTCAGGTTACCACGCGTAAAACATTACAC ADAM ACGCGGTTCAGGTTACCCCGCGTAGCGCATTACAC PAM The sequence homology between Individuals is not perfect!!! This allows us to assign a specific sequence to a specific Individual

  5. Homology (molecular biology) Regions of the DNA (gene or non-gene) that share similar nucleotide sequence Sequence homology is a very important concept Structural homology (nucleotide sequence) implies functional homology Genes with a similar sequence are likely to function in a similar manner Variation in sequence between individuals is also very Important

  6. Restriction Enzymes What are Restriction enzymes What are restriction enzyme RECOGNITION sites in DNA How do we map Restriction enzyme sites in DNA How do we use restriction enzymes to clone pieces of DNA How do we use restriction enzyme sites/maps to study individuals

  7. Restriction Enzymes Enzymes which Recognize a SPECIFIC DNA sequence BIND that sequence and CUT the DNA at that specific sequence SmaI is a Restriction enzyme | 5’ AAAACCCGGGAAAA3’ 3’ TTTTGGGCCCTTTT5’ | This sequence is symmetrical. If one rotates it about the axis It reads the same EcoRI is another Restriction enzyme that recognizes the sequence and cuts the sequence (but not in the middle) | 5’ AAAAGAATTCAAAA3’ 3’ TTTTCTTAAGTTTT5’ | Some restriction enzymes recognize a specific sequence that is 4 bp long Some restriction enzymes recognize a specific sequence that is 6 bp long Some restriction enzymes recognize a specific sequence that is 8 bp long

  8. BamHI Restriction enzymes Restriction enzyme digestion of DNA (linear genomic double stranded DNA) OR Restriction enzyme digestion of bacterial plasmid DNA (small double stranded circular DNA) No digestion of RNA No digestion of single stranded DNA

  9. Linear/Circular DNA No digestion of RNA No digestion of single stranded DNA A linear DNA molecule with ONE SmaI site will be cut into two fragments A circular DNA molecule with ONE SmaI site will generate one DNA fragment

  10. Blunt Vs Sticky Blunt ends Sticky ends After digestion of DNA by a restriction enzyme the DNA ends are either blunt or sticky

  11. Restriction sites SmaI- BLUNT ENDS 5’AAAAAAAAAAGGGGGGGGTTTTTTTCCCGGGAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAAGGGCCCTTTTTTTTCCCCCCCCAAAAAA5’ 5’AAAAAAAAAAGGGGGGGGTTTTTTTCCC GGGAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAAGGG CCCTTTTTTTTCCCCCCCCAAAAAA5’ EcoRI is another commonly used restriction enzyme 5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’ 5’AAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’ Unlike SmaI which produces a blunt end, EcoRI produces sticky or cohesive ends (SINGLE STRANDED) These cohesive ends facilitate formation of recombinant DNA molecules

  12. 5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’5’AAAAAAAAAAGGGGGGGGTTTTTTTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’ 5’AAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’ AATTCACGTACGTACGTACGTACGTACGTG GTGCATGCATGCATGCATGCATGCACTTAA 5’AAAAAAAAAAGGGGTTTTTTTG AATTCACGTACGTACGTACGTACGTACGTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTACCCCAAAAAAACTTAA GTGCATGCATGCATGCATGCATGCACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’ 5’AAAAAAAAAAGGGGTTTTTTTGAATTCACGTACGTACGTACGTACGTACGTGAATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTACCCCAAAAAAACTTAAGTGCATGCATGCATGCATGCATGCACTTAAGTTTTTTTTCCCCCCCCAAAAAA5’

  13. Complementary sticky ends AATTCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’ AAAAAAGGGGGGGGTTTTTTTG TTTTTTCCCCCCCCAAAAAAACTTAA AAAAAAGGGGGGGGTTTTTTTG TTTTTTCCCCCCCCAAAAAAACTTAA AATTCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’ GGCCCAAAAAAAAGGGGGGGGTTT3’ GTTTTTTTTCCCCCCCCAAA5’ AAAAAAGGGGGGGGTTTTTTTG TTTTTTCCCCCCCCAAAAAAACTTAA

  14. Enzyme compatibility SmaI AAACCCGGGAAA XmaI AAACCCGGGAAA TTTGGGCCCTTT TTTGGGCCCTTT EcoRI AAAGAATTCAAA MfeI AAACAATTGAAA TTTCTTAAGTTT TTTGTTAACTTT AAAGAATTGAAA TTTCTTAACTTT Cant be cut by EcoRI or MfeI KpnI AAAGGTACCAAA Asp718 AAAGGTACCAAA TTTCCATGGTTT TTTCCATGGTTT

  15. SmaI AAAAAACCCGGGAAAAAA ------------------ TTTTTTGGGCCCTTTTTT XmaI AAAAAACCCGGGAAAAAA ------------------ TTTTTTGGGCCCTTTTTT EcoRI AAAAAAGAATTCAAAAAA ------------------ TTTTTTCTTAAGTTTTTT MfeI AAAAAACAATTGAAAAAA ------------------ TTTTTTGTTAACTTTTTT KpnI AAAAAAGGTACCAAAAAA ------------------ TTTTTTCCATGGTTTTTT Asp718 AAAAAAGGTACCAAAAAA ------------------ TTTTTTCCATGGTTTTTT

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  17. Restriction maps Restriction maps are descriptions of the number, type and distances between Restriction sites on a piece of DNA. Very useful for molecular biologists. Previously we used specific genes as markers on chromosome and Map units to indicate distance between the markers. Its like using specific landmarks to identify your location along a road. Restriction sites are also used as landmarks along a piece of DNA. 11Mu 5Mu pr vg cy Vg Cy Pr 205kb 300kb 100kb PstI SmaI EcoRI HindIII NNNNNNNNNGAATTCNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNCTGCAGNNNNNNNNNNCCCGGGNNNNNN NNNNNNNNNCTTAAGNNNNNNNNNNNNTTCGAANNNNNNNNNNNNGACGTCNNNNNNNNNNGGGCCCNNNNNN AAAAAAAAAGAATTCTTTTTTTTTTTTAAGCTTCCCCCCCCCCCCCTGCAGGGGGGGGGGGCCCGGGAAAAAA TTTTTTTTTCTTAAGAAAAAAAAAAAATTCGAAGGGGGGGGGGGGGACGTCCCCCCCCCCCGGGCCCTTTTTT Restriction sites CAN serve as MARKERS ALONG the DNA. They can be used to generate a physical map of a specific DNA sequence can be created.

  18. Restriction maps Human Genome is 1.5 billion basepairs long There are 25,000 genes (markers) Every gene is on average approximately 600,000 bp apart EcoRI sites are on average 4000 bp apart HinDIII sites are on average 4000 bp apart Etc etc There are many more Restriction enzyme sites (landmarks) on any one piece of DNA then there are genes (landmarks)

  19. Human Chimp Gibbon Sequence Divergence The restriction map is a partial picture of the nucleotide sequence of a piece of DNA. By comparing restriction maps we can surmise differences in the DNA sequence between species or between individuals NNNNNNNNGAATTCNNNNNNNNNNNNNNNNAAGCTTNNNNNNNNNNNNNNCTGCAGNNNNNNNNNNNNNNN NNNNNNNNCTTAAGNNNNNNNNNNNNNNNNTTCGAANNNNNNNNNNNNNNGACGTCNNNNNNNNNNNNNNN GeneA Mai California me rahta hu aur UCSC me padhata hu. Mai California me rahta hu aur UCSC and UCLA me padhata hu. Mai California me rahta hu aur mai sirf UCLA me padhta hu

  20. Deletions and additions Normal Globin gene 3 5 8 4 EcoRI EcoRI EcoRI HindIII HindIII Globin gene from a thallesimia patient 3 5 3 4 EcoRI EcoRI EcoRI HindIII HindIII With restriction maps, the relationship between a gene from two different individuals can be determined without having to actually sequence the gene from both individuals.

  21. Very Large deletions or insertions can be studied using microscopy Small to large deletions/insertions (100 bp to several kb) can be studied using restriction maps!!

  22. Restriction map Describing a DNA piece based on the pattern of restriction sites Restriction map of a cloned piece of DNA Linear or circular H E E B K S B B B K S How do I do this for an unknown piece of DNA?

  23. Analogy 1 Goal: Identify the parts for this blob and describe the spatial relationship between the parts 2 You first Break the blob apart into its separate parts. Look at the shapes and number of the parts a c a 3 Fit the parts together c b b

  24. Method To Restriction map of piece of DNA Large amount of pure DNA (many copies of the same DNA is required) (Cloned) Take (cloned) DNA in a tube, Add restriction enzyme, Allow enzyme to cut DNA at its binding sites Run the digested DNA on a gel to resolve the DNA fragments based on size Piece the fragments together to determine the linear order of the fragments (build the map)

  25. EcoRI/HindIII HindIII Marker EcoRI - + 1 3 5 2 EcoRI EcoRI EcoRI HindIII HindIII Gel electrophoresis Agarose gel electrophoresis The length of the DNA can be accurately determined by allowing the charged DNA to run through an agarose gel. DNA is an anion (-ve charged) and moves towards the Positive anode. The rate of migration of a DNA fragment is inversely proportional to its size. Larger the size, slower its movement. EcoRI/HindIII HindIII Marker EcoRI 7 5 4 3 2 1

  26. 1 3 5 2 EcoRI EcoRI EcoRI HindIII HindIII EcoRI/HindIII HindIII Marker EcoRI 7 Cut with EcoRI 5 4 1 8 3 2 2 Cut with HindIII 1 7 4 Cut with EcoRI and HindIII 1 3 5 2

  27. 12 6 2 Mapping unknown fragment You are given a 20 kb linear fragment of DNA After trying many enzymes you find that EcoRI and HindIII cut the fragment HindIII 14kb and 6kb EcoRI 12kb 6kb and 2kb Solve the map HindIII Marker EcoRI uncut H 6 Or its mirror image 14 20 14 12 6 14 They are the same 6 4 What about EcoRI? 2 14 6 1 6 12 2 6 2 12 2 6 12 How do you arrange these three fragments wrt one another

  28. Mapping Since HindIII cut the 20kb fragment once, in which of the three EcoRI fragment does it cut? A double digest with both enzymes will provide the answer Fragments of 8kb, 6kb, 4kb and 2kb The double digest does not alter the size of the 6kb and 2kb fragments The 12kb fragment is lost. Also 8+4=12 So HindIII site must be in the 12 kb EcoRI fragment 2 6 EcoRI+HindIII 12 HindIII Marker EcoRI H H H 6 6 6 2 2 2 8 8 8 4 4 4 14 12 8 6 4 4 2 1 HindIII alone gives 14 & 6 Which of the three maps gives 14 and 6 for HindIII alone?

  29. New Mapping How are these fragments ordered? The HindIII single digest tells us that they must be ordered so that One side adds up to 6kb and the other side adds up to 14kb EcoRI+ HindIII HindIII Marker EcoRI 14 12 6 4 2 1

  30. Mapping HindIII EcoRI HindIII/EcoRI 14 12 8 6 6 6 4 2 2 HindIII 4 8 12 & 8 6 2 12 4 8 16 & 4 6 2 12 4 8 10 & 10 6 2 12 4 8 14 & 6 6 2 12 4 8 12 & 8 6 2 12 4 8 16 & 4 6 2 12

  31. Another linear map EcoRI+ HindIII PstI EcoRI+ PstI HindIII Marker EcoRI 14 12 8 6 4 2 1

  32. Different Mapping example Hi Ec Hi/Ec 12 12 8 8 6 6 4 2 2 Ps Ps/Ec 13 12 7 5 2 1 Three different enzymes Hi Ec Ps

  33. Mapping HindIII EcoRI HindIII/EcoRI 12 12 8 8 6 6 4 2 2 HindIII 16 H 8 4 16 & 4 6 2 12 E E 12 H 4 8 12 & 8 6 2 12 E E 12 H 4 8 12 & 8 2 6 12 E E

  34. Mapping EcoRI PstI PstI/EcoRI 12 13 12 6 7 5 2 2 1 6 2 12 P Pst I 1 5 5 & 15 6 2 12 E E P 1 5 1 & 19 6 2 12 E E 2 6 12 E E 1 5 P 3 & 17 2 6 12 E E 1 5 P 7 & 13 2 6 12 E E 4 8 H

  35. Final Map P 5 1 8 4 2 E E H Hi Ec Hi/Ec 12 12 8 8 6 6 4 2 2 Ps Ps/Ec 13 12 7 5 2 1

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  37. THE GENE PROBE!!! How do we isolate a GENE? How did we get a pure copy of the gene?

  38. Cloning DNA A reasonable question is how did we clone a fragment of DNA Or how do we clone a gene The construction of Recombinant DNA molecules or cloning of DNA molecules Recombinant DNA is generated through cutting and pasting of DNA to produce novel sequence arrangements Restriction enzymes such as EcoRI produce staggered cuts leaving short single-stranded tails at the ends of the fragment. These “cohesive or sticky” ends allow joining of different DNA fragments | nnnGAATTCnnn nnnCTTAAGnnn | When a piece of DNA is cut with EcoRI, you get nnnG AATTCnnn nnnCTTAA Gnnn

  39. Cloning DNA A reasonable question is how did we get the 20kb fragment of DNA in the first place? To understand the origin of the fragment we must address the issue of: The construction of Recombinant DNA molecules or cloning of DNA molecules Recombinant DNA is generated through cutting and pasting of DNA to produce novel sequence arrangements

  40. Recombinant DNA Restriction enzymes such as EcoRI produce staggered cuts leaving short single-stranded tails at the ends of the fragment. These “cohesive or sticky” ends allow joining of different DNA fragments | GAATTC CTTAAG | When a piece of DNA is cut with EcoRI, you get AATTCTTTTTTTTTTTAAAAAAGAATT GAAAAAAAAAAATTTTTTCTTAA AATTCAAAAAGGGGGTTTTTTTG TTAAGTTTTTCCCCCAAAAAAACTTAA 5’AAAAAAAAAAGGGGTTTTTTTG AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTACCCCAAAAAAACTTAA GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’

  41. Plasmids Plasmids are naturally occurring circular pieces of DNA in E. coli The plasmid DNA is circular and usually has one EcoRI site. It is cut with EcoRI to give a linear plasmid DNA molecule AATT AATT

  42. Small circular autonomously replicatingextrachromosomal DNA Modified plasmids, called cloning vectors are used by molecular biologists to isolate large quantities of a given DNA fragment Plasmids used for cloning share three properties Unique restriction site Antibiotic resistance Origin of replication Plasmids B E Bacterial genome (5000kb) Plasmid DNA (3kb) Origin Antibiotic resistance gene

  43. Plasmid elements Origin of replication: This is a DNA element that allows the plasmid to be replicated and duplicated in bacteria. Each time the bacterium divides, the plasmid also needs to divide and go with the daughter cells. If a plasmid cannot replicate in bacteria, then it will be lost.

  44. Plasmid elements Antibiotic resistance: This allows for the presence of the plasmid to be selectively maintained in a given strain of bacteria +antibiotics -antibiotics Lab bacterial strains are sensitive to antibiotics. When grown on plates with antibiotics, they die. The presence of a plasmid with the antibiotics resistance gene allows these lab strains to grow on plates with the antibiotic. You are therefore selecting for bacterial colonies with the Plasmid

  45. Plasmid elements Unique restriction sites: For cloning the plasmid needs too be linearized. Most cloning vectors have unique restriction sites. If the plasmid contains more than one site for a given restriction enzyme, this results in fragmentation of the plasmid Why does this matter? Antibiotic resistance gene Ori

  46. pUC18 pUC18 is one of the most commonly used plasmid: pUC= plasmid University of California Plasmid replicon copy No pBR322 pMB1 15 pUC18 pMB1 500 pACYC p15A 10 pSC101 pSC101 5

  47. Cloning DNA | GAATTC CTTAAG | When a piece of DNA is cut with EcoRI, you get 5’AAAAAAAAAAGGGGTTTTTTTG AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG AATTCAAAAAAAAGGGGGGGGTTTTTT3’ 3’TTTTTTTTTACCCCAAAAAAACTTAA GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA GTTTTTTTTCCCCCCCCAAAAAA5’ AATTCAAAAAAAAAAAAAAGGGGGGGGTTTTTTTG GTTTTTTTTTTTTTTCCCCCCCCAAAAAAACTTAA 5’AAAAAAAAAAGGGGTTTTTTTG 3’TTTTTTTTTACCCCAAAAAAACTTAA When two pieces of DNA cut with EcoRI are ligated back together you get back an EcoRI site -----------------G -----------------CTTAA AATTC----------------- G-----------------

  48. AATT PLASMID TTAA AATT AATT TTAA AATT TTAA TTAA TTAA TTAA TTAA TTAA TTAA TTAA TTAA TTAA Ligation GENOMIC DNA TheEcoRI linearized PLASMID DNA is mixed with HUMAN DNA digested with EcoRI The sticky ends will hybridize/anneal specifically and a recombinant plasmid will be generated

  49. Ampr Ori d E F G h Cloning The genomic DNA fragments is mixed with a plasmid that has been linearized at a single EcoRI site (say pUC18) Both the plasmid and genomic DNA have been cut with EcoRI, they have complementary sticky ends | G A A T T C C T T A A G | ________________________ ________________________TTAA AATT----------------------------- -----------------------------TTAA AATT Genomic DNA Plasmid

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