1

2/16/2022

Chapter Five: Exploring Genes and Genomes

2

2/16/2022

DNA Sequencing

​

Polymerase Chain Reaction

​

DNA Recombination Technology

siRNA

Topics

Introduction

Application

Reactions

Process

Definition

Importance

Requirements and how

Introduction

Requirements and how

3

2/16/2022

• Sanger sequencing = Dideoxy sequencing = Chain termination sequencing;
• This is a method pioneered by Fred Sanger. Frederick Sanger obtained his second time Nobel Prize in 1980;
• Each of these refers to the same method;
• The use of an enzyme, known as DNA polymerase, to replicate a polynucleotide;
• The occasional incorporation of a dideoxy base in the newly produced DNA strand, with resultant termination of that individual DNA strand.

DNA sequencing

4

2/16/2022

• Dideoxy sequencing can sequence ~800 bp/run.
• Human genome project was completed in 2003. It took 13 years.
• Next generation sequencing was developed by biotechnology companies, e.g. ABI, Illumina and Roche. This new technology can sequence ~10⁸ bp/run.

DNA sequencing

• A template: a single stranded piece of DNA that we are going to replicate. It is by necessity a single strand (rather than "double helix", or double strand) because in order to replicate the DNA we need access to the bases in the center of the duplex. The only way we can get access is to separate the strands of the duplex. We will work with one of the strands. Since the other strand in the duplex is complementary (A pairs with T, and G pairs with C) if we know the sequence of one strand, we can infer the sequence of the other in the duplex, so we really only need to sequence one strand.
• A primer: DNA polymerase enzyme can only extend an existing fragment of DNA that is complementary to the template. It can't actually start making a complementary strand. Thus, we need a short piece of DNA that is complementary to some starting point in the template (and will hydrogen-bond and bind to form a duplex); Short RNA in vivo during DNA replication.
• dNTP bases: deoxynucleside triphosphates are used to build the new DNA strand. The high energy phosphate bond in these nucleotides will provide the energy needed for synthesis - we don't need to add anything else for this energy.
• DNA polymerase: all polymerases go in the same direction. They synthesize a new polynucleotide strand in the 5‘ to 3' direction. Due to the anti-parallel nature of the two strands in a DNA duplex, this means that the polymerase is actually moving in the 3‘ to 5' direction with regard to the template DNA. What we also need for Sanger/di-deoxy/chain-termination sequencing.
• ddNTP bases: at the heart of this method is the occasional incorporation of a dideoxy base. So we need ddNTP bases. The "dideoxy" means that the bases have hydrogens, instead of hydroxyls, at both the 2' and 3' ribose positions.

5

2/16/2022

Fundamental principles of DNA replication by a DNA polymerase

6

2/16/2022

DNA polymerase copies ssDNA in vitro in the presence of the four deoxynucleotide monomers, provided a double-stranded region of DNA is artificially generated by adding a primer, an oligonucleotide capable of forming a short stretch of dsDNA by base pairing with the ssDNA. The primer must have a free 3'-OH end from which the new polynucleotide chain can grow as the first residue is added in the initial step of the polymerization process.

7

2/16/2022

8

2/16/2022

dNTPs

ddNTPs

9

2/16/2022

Reaction A

5’-GCTACGCTCTGA-3’

ddATGCGAGACT-5’

ddATP

dNTP (dATP,dGTP,dCTP,dTTP)

ddAGACT-5’

ddATGCGAGACT-5’

1

6

0.5 ml Eppendorf tube

primer

known sequence

to be determined

10

2/16/2022

5’-GCTACGCTCTGA-3’

ddGATGCGAGACT-5’

Reaction G

ddGTP

dNTP(dATP,dGTP,dCTP,dTTP)

ddGAGACT-5’

ddGCGAGACT-5’

ddGATGCGAGACT-5’

2

4

7

11

2/16/2022

5’-GCTACGCTCTGA-3’

GACT-5’

Reaction C

ddCTP

dNTP(dATP,dGTP,dCTP,dTTP)

ddCGAGACT-5’

ddCGATGCGAGACT-5’

3

8

12

2/16/2022

5’-GCTACGCTCTGA-3’

ddTGCGAGACT-5’

Reaction T

ddTTP

dNTP(dATP,dGTP,dCTP,dTTP)

ddTGCGAGACT-5’

5

13

2/16/2022

A

5’-GCTACGCTCTGA-3’

GACT-5’

ddATP

dNTP

G

ddGTP

dNTP

C

ddCTP

dNTP

T

ddTTP

dNTP

ddAGACT-5’

ddATGCGAGACT-5’

ddGAGACT-5’

ddGCGAGACT-5’

ddGATGCGAGACT-5’

ddCGAGACT-5’

ddCGATGCGAGACT-5’

ddTGCGAGACT-5’

1

2

3

4

5

6

7

8

14

2/16/2022

A

5’-GCTACGCTCTGA-3’

CGATGCGAGACT-5’

ddATP

dNTP

G

ddGTP

dNTP

C

ddCTP

dNTP

T

ddTTP

dNTP

ddAGACT-5’

ddATGCGAGACT-5’

ddGAGACT-5’

ddGCGAGACT-5’

ddGATGCGAGACT-5’

ddCGAGACT-5’

ddCGATGCGAGACT-5’

ddTGCGAGACT-5’

1

2

3

4

5

6

7

8

A G C T

A

G

C

G

T

A

G

C

To be determined

1

2

3

4

5

6

7

8

15

2/16/2022

16

2/16/2022

Human genome: ~3 x 10⁹ bp

17

2/16/2022

DNA Sequencing

​

Polymerase Chain Reaction

​

DNA Recombination Technology

siRNA

Topics

Introduction

Application

Reactions

Process

Definition

Importance

Requirements and how

Introduction

Requirements and how

18

2/16/2022

Introduction

• Polymerase Chain Reaction (PCR) provides an extremely sensitive means of amplifying relatively large quantities of DNA;
• First described in 1985, Nobel Prize for Kary Mullis in 1993;
• The technique was made possible by the discovery of Taq polymerase (optimum temperature is 75-80ºC, the DNA polymerase that is used by the bacterium Thermus aquaticus that was discovered in hot springs (Yellow Stone National Park).

19

2/16/2022

Application

• Amplify genes;
• PCR-based mutagenesis;
• The amplified DNA from cells can be used in DNA fingerprinting analysis to determine who was at the crime scene;
• Diagnosis of genetic diseases;
• RT-PCR (reverse transcriptase) and real-time PCR: gene expression.

20

2/16/2022

Requirements of Polymerase Chain Reactions

• DNA nucleotides (dNTP);
• Template DNA: the DNA sequence that you want to amplify (gDNA library, cDNA library, DNA fragments, plasmids);
• Primers: two single-stranded DNAs between 20 and 50 nucleotides;
• Taq DNA polymerase: a heat stable enzyme that drives, or catalyzes, the synthesis of new DNA.

21

2/16/2022

Three Major Steps in a PCR Reaction

• Denaturation at around 94°C: During the denaturation, the double strands melt open to single stranded DNA, all enzymatic reactions stop (for example the extension from a previous cycle);
• Annealing at around 55°C: Hydrogen bonds are constantly formed and broken between the single stranded primer and the single stranded template. If the primers exactly fit the template, the hydrogen bonds are so strong that the primer stays attached;
• DNA synthesis at around 65°C: The bases (complementary to the template) are coupled to the primer on the 3' side (the polymerase adds dNTP's from 5' to 3', reading the template from 3' to 5' side, bases are added complementary to the template).

22

2/16/2022

Genome

23

2/16/2022

Denaturation

Annealing

Sense primer

Denaturation

Annealing

DNA synthesis

Denaturation

Annealing

DNA synthesis

Cycle 0

Number of Copy

1

Cycle 1

2

Cycle 2

4

Cycle 3

8

Antisense primer

DNA synthesis

Cycle 1

Cycle 2

Cycle 3

Cycle 35

Cycle 40

= 2⁰

= 2¹

= 2²

= 2³

2³⁵

2⁴⁰

24

2/16/2022

Problem 1: If we start with 10 copies of genomic dsDNA, how many copies do you have after 35 cycles of PCR?

Problem 2: If we start with 10 copies of ssDNA, how many copies do you have after 35 cycles of PCR?

10 x 2³⁵

10 x 2³⁴

25

2/16/2022

5’-AGTCTTAAAGTCTAAA-3’

3’-TCAGAATTTCAGATTT-5’

5’-AGT-3’

Sense primer

3’-TTT-5’

Antisense primer

3’-TCAGAATTTCAGATTT-5’

5’-AGT-3’

Sense primer

3’-TTT-5’

Antisense primer

5’-AGTCTTAAAGTCTAAA-3’

26

2/16/2022

5’-AGTCTTAAAGTCTAAA-3’

5’-AGT-3’

Sense primer

3’-TTT-5’

Antisense primer

27

2/16/2022

DNA Sequencing

​

Polymerase Chain Reaction

​

DNA Recombination Technology

siRNA

Topics

Introduction

Application

Reactions

Process

Definition

Importance

Requirements and how

Introduction

Requirements and how

DNA Recombinant

DNA recombination is a technology that allows a gene or a DNA fragment to transfer from one species to another.

28

2/16/2022

An example to show the importance of DNA recombinant technology

29

2/16/2022

Pharmaceutical company: Eli Lilly: Humalog (human insulin)

30

2/16/2022

• 100 millions diabetes patients world wide
• Each person: one vial insulin/month = Humalog 100 units/ml (equivalent to 3.5 mg)
• Total amount needed each year world wide: ~40 mg/person and year x 100 x 10⁶ = 4.0 x 10⁹ mg = 4.0 x 10⁶ g
• How to produce 4 million gram of insulin? From human blood, or cow blood? Impossible!
• A typical blood level between meals is 8–11 μIU/mL (57–79 pmol/L)

Background Information on Insulin

Eli Lilly and Company Limited

Examples: Recombinant Human Insulin

31

2/16/2022

Requirements: �DNA Recombinant Technology

32

2/16/2022

• Restriction enzymes (restriction endonucleases Type II)
• Cloning vectors
• Sources of target genes or fragments (gDNA or cDNA library)
• Gene amplification (PCR)
• Expression system
• DNA ligases

33

2/16/2022

Nucleases

• Enzymes that hydrolyze nucleic acids are called nucleases.
• Nucleases are located in lysosome and proteasome inside eukaryotic cells. They functions in degradation of nucleic acids, DNA repair.

34

2/16/2022

Enzymatic Hydrolysis

Exonucleases

Endonucleases

Nuclease a

Nuclease b

DNase

RNase

Cleave at end or middle

Cleave at right or left side of Pi

Nucleic acid specificity

Nucleases

ds

ss

Strand specificity

35

2/16/2022

Enzymatic Hydrolysis

• Exonucleases start at the end of a polynucleotides (either the 3' or 5' end, depending on their specificity) and sequentially hydrolyze the phosphodiester bond in the polynucleotide.
• Endonucleases cleave at an internal site within the polynucleotide. Although there may be multiple internal sites that are susceptible, the endonucleases do not subsequently sequentially hydrolyze the phosphodiester bond at such sites.

36

2/16/2022

Nucleases Can Cleave on Either Side of the Phosphodiester Bond

• Type "A" nucleases cleave on the 3' side of the phosphodiester bond. Such a cleavage leaves the phosphate on the 5' side of the leaving polynucleotide;
• Type "B" nucleases cleave on the 5' side of the phosphodiester bond. Such a cleavage leaves the phosphate remaining on the 3' end of the polynucleotide;
• Nucleases can also exhibit hydrolysis specificity towards single stranded polynucleotides (DNA or RNA), or duplex (i.e. double helix) polynucleotides (DNA is typically the only duplex to be concerned about).

37

2/16/2022

Nucleases Can Cleave on Either Side of the Phosphodiester Bond

38

2/16/2022

P

P

P

P

OH

P

A

G

C

T

G

Enzyme A

Enzyme B

Enzyme A: exonuclease

Enzyme A: nuclease a

Enzyme A: DNase

Enzyme A: ss nuclease

Enzyme B: endonuclease

Enzyme B: nuclease b

Enzyme B: DNase

Enzyme B: ss nuclease

39

2/16/2022

Restriction Endonucleases

Restriction endonucleases or called restriction enzymes are produced by microorganisms as part of the defense system to protect themselves against invasion of foreign DNA by cleaving double stranded DNAs.

40

2/16/2022

Type I, II and III Restriction Endonucleases

• Type I: requires ATP for hydrolysis; is also able to methylate DNA at specific locations; cuts at random sites;
• Type III: requires ATP for hydrolysis; is also able to methylate DNA at specific locations; cuts at specific nucleotide sequences;
• Type II: does not require ATP for hydrolysis (uses energy within phosphodiester bond for hydrolysis); do not methylate DNA; cut at specific sites; typically cut at a palindromic sequence in duplex DNA; can leave DNA duplex ends that are either "blunt", or "sticky”; sticky ends can be "3' overhang" or "5' overhang“; sticky ends of different DNA fragments produced by the same enzyme are complementary (and can be joined together in new ways).

41

2/16/2022

42

2/16/2022

Restriction Enzymes

DNA ligase can seal it.

GATATC

CTATAG

EcoR V

ACCGGT

TGGCCA

AGCGCT

43

2/16/2022

G

CCTAG

GATCC

G

GGATCC

CCTAGG

BamH I

Sticky end

GAT

CTA

ATC

TAG

GATATC

CTATAG

EcoR V

Blunt end

Requirements: �DNA Recombinant Technology

44

2/16/2022

• Restriction enzymes (restriction endonucleases Type II)
• Cloning vectors
• Sources of target genes or fragments (gDNA or cDNA library)
• Gene amplification (PCR)
• Expression system
• DNA ligases

Types of Vectors

• Plasmids: <10 kb;
• Bacteriophage lambda: <16 kb;
• Cosmids: 40 kb;
• BAC (bacterial artificial chromosome): 150-300 kb;
• YAC (yeast artificial chromosome): <2 x 10⁶ bp
• Shuttle vectors: E.coli or yeast

45

2/16/2022

Plasmids

46

2/16/2022

Plasmids are naturally occurring extrachromosomal circular dsDNA.

​

What are plasmids?

A bacterial cell

Genome

Plasmids

Features of Plasmids

• Origin of replication (ori);
• Multiple cloning sites: restriction endonucleases;
• A selection marker: drug resistance (ampicillin, kanamycin, spectinomycin and chloramphenicol)

47

2/16/2022

48

2/16/2022

49

2/16/2022

Ampicillin

50

2/16/2022

Bacterial cell wall

Transpeptidase

Transpeptidase

Active

Transpeptidase

Inactive

51

2/16/2022

What are the difference between Amp and Amp^R?

52

2/16/2022

Thiazolidine ring

Lactam ring

Amp^R gene

β-Lactamase

Ampicillin

Ampicillin is the substrate of Amp^R enzyme (β-Lactamase).

Ampicillin: An Antibiotic Drug

• Ampicillin binds to and inhibits a number of enzymes, especially transpeptidase in the bacterial membrane that are involved in the synthesis of the gram negative cell wall;
• Addition of ampicillin to the growth media causes faulty cell wall production in E. coli and this causes problems particularly during cell replication;
• Proper cell replication cannot occur in the presence of ampicillin and the E. coli will die (hence, ampicillin is an antibiotic);
• The chemical structure of ampicillin contains a β-lactam ring;
• The ampicillin resistance gene (amp^R) codes for an enzyme (β-lactamase) that is secreted into the periplasmic space of the bacterium where it catalyzes hydrolysis of the β-lactam ring of the ampicillin;
• Hydrolysis the β-lactam ring in the ampicillin destroys its function;
• The enzyme product of the amp^R gene destroys the antibiotic.

53

2/16/2022

Antibiotics

• Kanamycin: binds and inhibits 30S subunit of prokaryotic ribosome (Prokaryotic ribosome: 30S and 50S subunits, and Eukaryotic ribosome: 40S and 60S subunits).
• Spectinomycin: binds and inhibits 30S subunit of prokaryotic ribosome.
• Chloramphenicol: binds and inhibits 50S subunit of prokaryotic ribosome.

54

2/16/2022

55

2/16/2022

DNA Ligase

56

2/16/2022

57

2/16/2022

Requirements: �DNA Recombinant Technology

58

2/16/2022

• Restriction enzymes (restriction endonucleases Type II)
• Cloning vectors
• Sources of target genes or fragments (gDNA or cDNA library)
• Gene amplification (PCR)
• Expression system
• DNA ligases

Genomic DNA Library

• A collection of cloned DNA fragments that together represent all the genes of a particular organism;
• Plasmids can reliably maintain and replicate a DNA fragment of up to 10 kb;
• Fragmentation of a genome can be accomplished using an appropriate restriction endonuclease. Use a 6-cutter for an average fragment size of 4 kb. Use an 8-cutter for an average fragment size of 65 kb;
• Open the plasmid using the same restriction enzyme and the fragments are cohesive with the linearized plasmid;
• Ligate using DNA ligase;
• Each plasmid gets on average one fragment.

59

2/16/2022

cDNA Libraries

• Another type of library is a cDNA library (where the "c" means "complementary");
• A cDNA library starts with the mRNA extracted from a specific tissue or cell type from an organism;
• The mRNA is converted "back" into its complementary DNA using an RNA-dependent DNA polymerase (from a virus);
• The duplex DNA thus produced is subcloned into a plasmid library;
• The unique feature of a cDNA library is that it contains the genetic information for expressed genes (i.e. those that are producing mRNA for protein production) and that they are from a specific tissue;
• cDNA libraries from different tissues of identical organisms will be different. Also, cDNA libraries from the same tissue, but different developmental stages (e.g. embryo versus adult) will be different. So will cDNA libraries from identical organisms with the same age and tissue, but with different diseased states.

60

2/16/2022

Making cDNA

61

2/16/2022

• An extremely useful source of DNA since RNA is unstable and cannot be cloned directly;
• Especially useful in analyzing eukaryotic gene – why?
• Tissue specific cDNA

or RNase H

Requirements: �DNA Recombinant Technology

62

2/16/2022

• Restriction enzymes (restriction endonucleases Type II)
• Cloning vectors
• Sources of target genes or fragments (gDNA or cDNA library)
• Gene amplification (PCR)
• Expression system
• DNA ligases

Examples: Recombinant Human Insulin

63

2/16/2022

• mRNA of human insulin;
• cDNA of human insulin (cDNA, complementary DNA) library);
• PCR-amplified cDNA;
• Restriction enzyme cleavage;
• DNA ligation

ligase

insulin

vector

64

2/16/2022

Expression System

(Plasmid)

Human insulin cDNA

Bacterial promoter

Bacterial terminator

Bacterial ribosome binding sequence

65

2/16/2022

DNA Sequencing

​

Polymerase Chain Reaction

​

DNA Recombination Technology

siRNA

Topics

Introduction

Application

Reactions

Process

Definition

Importance

Requirements and how

Introduction

Requirements and how

66

2/16/2022

Gene expression inhibited by siRNA

mRNA needs to be degraded

siRNA

Small mRNA fragments

67

2/16/2022

�

Dicer contains two RNase III domains

siRNAs

long dsRNA

Inverted repeat structures, bidirectional transcription and antisense transcripts from various loci are sources of the dsRNAs

�

68

2/16/2022

• Multidomain proteins with RNase activity
• about 200 kDa
• Evolutionary highly conserved
• Occurs in a wide range of eukaryotes, including protozoa, invertebrates and plants (in prokaryotes → RNase III)
• Involved in RNAi pathway (for gene-silencing)
• Dicer was given its name by Emily Bernstein, a graduate student in Greg Hannon's lab at Cold Spring Harbor Laboratory, who first demonstrated the enzyme's dsRNA "dicing" activity (Nature, 2001, 409 (6818): 363–6).

What are Dicer and where do they occur?

69

2/16/2022

siRNAs have a defined structure

70

2/16/2022

• siRNAs, ~20-25 bp long with a two-base overhang on the 3' end, are produced through dsRNA cleaved by Dicer;
• One strand of siRNA specifically binds its mRNA mediated by RISC, RNA-induced silencing complex;
• Binding of siRNA and RISC to a particular mRNA activates RISC and causes mRNA degradation by RISC.

Gene Silencing by siRNA

71

2/16/2022

• Dicer binds dsRNA.

​

• Dicer is an endoribonuclease in the RNase III family that cleaves dsRNA and pre-miRNA into short double-stranded RNA fragments about 20-25 bp long, with a two-nucleotide overhang on the 3' end.

​

• Dicer contains two RNase III domains and one PAZ domain; the distance between these two regions of the molecule is determined by the length and angle of the connector helix and influences the length of the siRNAs it produces.

72

2/16/2022

• The RNA-induced silencing complex (RISC) is a multiprotein complex that incorporates one strand of a small interfering RNA (siRNA) or microRNA (miRNA).

​

• RISC uses the siRNA or miRNA as a template for recognizing complementary mRNA. When it finds a complementary strand, it activates RNase and cleaves the RNA. RISC is in its inactive form when RISC binds ss siRNA or ss miRNA.

​

• This process is important both in gene regulation by miRNAs and in defense against viral infections, which often use double-stranded RNA as an infectious vector.

73

2/16/2022

DNA Sequencing

​

Polymerase Chain Reaction

​

DNA Recombination Technology

siRNA

Topics

Introduction

Application

Reactions

Process

Definition

Importance

Requirements and how

Introduction

Requirements and how

74

2/16/2022

Thank you for your attention!

Have a good day!

75

2/16/2022

• Learning is life-time experience!
• Learning chemistry from doing chemistry.
• A mistake is acceptable. The most important is “Learn something from each mistake and try not make the same mistake twice.”

Suggestions for Thinking

76

2/16/2022

Suggestions for Thinking

“The whole purpose of education is to turn mirrors into windows.” ~Sydney J. Harris

77

2/16/2022

Suggestions for Thinking

“It is the supreme art of the teacher to awaken joy in creative expression and knowledge.” ~Albert Einstein

78

2/16/2022

siRNA and miRNA are slightly different. siRNA can typically trigger more efficient and specific gene silencing; whereas one miRNA may compromise the expression of many different target genes simultaneously. Hence, siRNA and miRNA have different roles in pharmaceutical practice.