essential biochemistry - stfxwater plays a role in dictating biomolecular structure via the...
TRANSCRIPT
![Page 1: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/1.jpg)
Lecture Notes for
Chapter 3 From Genes to Proteins
Essential Biochemistry Third Edition
Charlotte W. Pratt | Kathleen Cornely
Copyright © 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 2: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/2.jpg)
KEY CONCEPTS: Section 3-1
• DNA and RNA are polymers of nucleotides, each of which consists of a: – Purine or pyrimidine base
– Deoxyribose or ribose
– Phosphate
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 3: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/3.jpg)
DNA and RNA have four nitrogenous bases.
• Purines: 2 examples • Pyrimidines: 3 examples • Notice the similarities in structure. • Notice differences in numbering around ring.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 4: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/4.jpg)
Adenine and guanine are purines in both DNA and RNA.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 5: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/5.jpg)
Learn how to draw purines. Adenine, A
• Draw ring system • Two more steps
– Add amino group (C6) – Fill in bonds
Guanine, G • Draw ring system • Three more steps
– Add amino group (C2) – Add oxo group (C6) – Fill in bonds
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 6: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/6.jpg)
Cytosine and thymine are pyrimidines found in DNA.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 7: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/7.jpg)
Cytosine and uracil are pyrimidines found in RNA.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 8: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/8.jpg)
Learn how to draw pyrimidines. Cytosine, C
• Draw ring system
• Two more steps – Add amino group (C4) – Fill in bonds
Uracil, U and Thymine, T • Draw ring system
• Two, three more steps – Add oxo group(C4) – Add H on N3 – Add methyl group (C5)
for thymine
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 9: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/9.jpg)
DNA and RNA contain sugar groups. Found in RNA Found in DNA
Notice: Ring numbering
on the sugars contain primes!
Ribose sugars have 2’-OH groups, while deoxyribose sugars lack a 2’-OH. © 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 10: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/10.jpg)
Phosphates can attach to the sugar at C3’ and/or C5’.
Monophosphates are shown in examples here. There can be up to 3 phosphates at a given terminus.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 11: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/11.jpg)
Nucleoside/nucleotide nomenclature is distinct from that of bases.
• Nucleoside = base + sugar
• Nucleotide = base + sugar + phosphate
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 12: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/12.jpg)
Nomenclature Summary Base Nucleoside Nucleotide (example using monophosphates) Adenine Adenosine Adenosine Monophosphate Guanine Guanosine Guanosine Monophosphate Thymine Thymidine Thymidine Monophosphate Cytosine Cytidine Cytidine Monophosphate Uracil Uridine Uridine Monophosphate
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 13: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/13.jpg)
Some nucleotides have functions other than encoding genetic information.
Nucleotides can be fused with vitamins to form molecules that aid in enzyme-catalyzed reactions.
(vitamin)
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 14: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/14.jpg)
How do nucleotides link?
• Via sugar in phosphodiester backbone
• Read sequence of bases from 5’à 3’
• Nucleotides are connected via phosphodiester bonds.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 15: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/15.jpg)
Bases form specific hydrogen bonds.
A and T form 2 H-bonds.
G and C form 3 H-bonds.
Base pair width is similar within a given conformation
of DNA. © 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 16: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/16.jpg)
Chargaff’s rule gives a clue to base pairing.
• Chargaff’s rule: the amount of A+G = C+T.
• Base pairs contain a purine and a pyrimidine.
• A base pairs with T or U; G base pairs with C.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 17: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/17.jpg)
KEY CONCEPTS: Section 3-1
• A DNA molecule contains two antiparallel strands that wind around each other to form a double helix in which A and T bases in opposite strands, and C and G bases in opposite strands, pair through hydrogen bonding.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 18: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/18.jpg)
DNA forms a double helix.
Ball-and-stick representation
Space-filling representation with phosphate
backbone emphasized in
white.
Strands are antiparallel.
What drives DNA to form a double helical shape in 3D?
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 19: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/19.jpg)
Water plays a role in dictating biomolecular structure via the
hydrophobic effect.
The hydrophobic effect is the phenomenon by which nonpolar molecules aggregate to avoid contact with
hydrophilic molecules, particularly water.
From Chapter 2
Unfavorable Many H2O molecules
are ordered around the nonpolar molecules.
Preferred Fewer H2O molecules
are ordered around the nonpolar molecules.
As H2O molecules are freed, entropy
of the system increases!
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 20: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/20.jpg)
Axial View of DNA
Base pairs form the core of the double helix.
Phosphate backbone forms the periphery.
Double helical conformation of DNA is driven by entropic
forces that induce base stacking.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 21: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/21.jpg)
Viewing 3D Structures of Macromolecules
• Go to http://www.rcsb.org/pdb/home/home.do
• Search for a molecule of interest.
• Select its link (e.g. 355D).
• Select download files à PDB file (text).
• Open the file in molecular viewing freeware.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 22: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/22.jpg)
DNA is stabilized by different forces.
• Predominant force: hydrophobicity, base stacking and entropy
• Hydrogen bonding (in base pairs) • Ionic interactions
– Cations (e.g. Mg2+, Na+, K+) – Polyamines
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 23: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/23.jpg)
KEY CONCEPTS: Section 3-1
• Double-stranded nucleic acids are denatured at high temperatures.
• At lower temperatures, complementary
polynucleotides anneal.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 24: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/24.jpg)
DNA can denature (unfold). • Native state • Denatured
state
• Abs = 260 nm
• Tm = melting temperature
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 25: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/25.jpg)
DNA can renature (refold, anneal).
DNA’s ability to re-anneal is very important – in nature and in biochemical research!
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 26: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/26.jpg)
KEY CONCEPTS: Section 3-2
• The biological information encoded by a sequence of DNA is: – Transcribed to RNA – Then, translated into the amino acid sequence
of a protein
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 27: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/27.jpg)
What does it mean to go from genes to proteins?
• Genes are sequences of DNA. • Replication: copying DNA • Transcription: converting DNA into RNA • Reverse transcription: converting RNA into DNA • Translation: making proteins from an RNA
template © 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 28: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/28.jpg)
DNA replication is critical to life.
• In vivo – DNA must be copied in order to sustain life. – Excessive DNA replication can be indicative of
cancer.
• In vitro – Technology known as the “polymerase chain
reaction” or PCR has revolutionized researchers capability to study nucleic acids and genes.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 29: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/29.jpg)
Transcription of DNA is critical to life.
Cells cannot thrive if transcription is shut down!
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 30: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/30.jpg)
Transcription RNA polymerase (not shown here) unwinds and separates dsDNA at the position
where transcription occurs.
Notice that the RNA transcript is complementary to the antisense (noncoding) strand!
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 31: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/31.jpg)
There are three fundamental types of RNA.
• Messenger RNA: encodes for polypeptide sequences
• Transfer RNA: carries amino acids to ribosome
• Ribosomal RNA: aids in polypeptide synthesis
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 32: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/32.jpg)
tRNA is single-stranded and forms unique conformations.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 33: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/33.jpg)
Translation results in protein synthesis.
Translation occurs in the ribosome, which contains rRNA and many other proteins.
In translation, tRNA carries amino acids to the ribosome and
binds to its complement in the mRNA template.
Amino acids are dictated by the Genetic Code.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 34: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/34.jpg)
The genetic code is used to translate mRNA into an amino acid sequence.
Read mRNA sequence in the Genetic Code
by the position of 3 nucleotides.
Notice the redundancy!
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 35: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/35.jpg)
KEY CONCEPTS: Section 3-3
• The genomes of different species vary in size and number of genes.
• Genes can be identified by their nucleotide sequences.
• Analysis of genetic data can provide information about gene function and risk of disease.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 36: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/36.jpg)
Gene size is roughly correlated with organismal complexity.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 37: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/37.jpg)
KEY CONCEPTS: Section 3-4
• A DNA molecule can be sequenced or amplified by using DNA polymerase to make a copy of a template strand.
• Linking together DNA fragments produces recombinant DNA molecules that can be used: – To study gene function – To express genes in host organisms – To engineer genes for commercial and
therapeutic purposes
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 38: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/38.jpg)
DNA can be sequenced via the Sanger method.
ddNTPs lack a 3’-OH group
No more NTP’s
can attach.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 39: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/39.jpg)
Dideoxy DNA Sequencing – Sanger Method
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 40: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/40.jpg)
Dideoxy DNA Sequencing – Sanger Method
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 41: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/41.jpg)
DNA can be amplified or copied using DNA polymerase.
• A process called the “Polymerase Chain Reaction” or PCR can be used to make billions of copies of DNA efficiently and accurately.
• PCR was developed by Kary Mullis, who won the 1993 Nobel Prize in Chemistry for his discovery.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 42: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/42.jpg)
What is required to make PCR work? • Heat-stable DNA polymerase (e.g., “Taq”
DNA polymerase)
• Primers (DNA oligonucleotides)
• Deoxynucleotide triphosphates
– dATP, dGTP, dCTP, dTTP
• DNA template
• Buffer containing Mg2+, among others
Also required:
Thermal Cycler – a device that heats and cools samples with speed, precision, and
reproducibility.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 43: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/43.jpg)
PCR repeats three chemical reactions.
• Step 1: Denaturation 92–95°C – dsDNA separates at high temp to form ssDNA.
• Step 2: Annealing ~55°C – Primers can base pair to ssDNA.
• Step 3: Extension 72°C
– Optimal temp for heat-stable DNA polymerases to work
– New strand is synthesized. © 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 44: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/44.jpg)
PCR is conducted over many cycles until millions of
copies are produced.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 45: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/45.jpg)
Restriction enzymes’ role in nature is to aid in defense for bacteria.
• Bacteria methylate their own DNA
• Bacteriophages (viruses that infect bacteria) have unmethylated DNA.
• Bacterial restriction enzymes can recognize and excise viral DNA.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 46: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/46.jpg)
Type II restriction enzymes are used to selectively cut DNA.
Type Activity Cleavage Site I Endonuclease & ≥ 1000 bp from
Methylase recognition sequence II Endonuclease Within or near
recognition sequence III Endonuclease & ~24-26 bp from
Methylase recognition sequence
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 47: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/47.jpg)
EcoRV 5’-G-A-T-A-T-C-3’ 3’-C-T-A-T-A-G-5’
EcoRI 5’-G-A-A-T-T-C-3’ 3’-C-T-T-A-A-G-5’
Characteristics of Type II Restriction Enzymes
• Cleave 4-8 bp segment of dsDNA
• Palindromic recognition sequences – “Madam, I’m Adam” – “Race Car”
Remember: Always read from
5' à 3'
Axis of symmetry
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 48: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/48.jpg)
Type II Restriction enzymes form blunt or sticky ends.
• EcoRI recognizes 5'-GAATTC-3'. • Complimentary sequence is implied. • EcoRI cuts both strands after the 5'-G. • Each fragment has sticky ends.
5' 5' 3' 3'
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 49: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/49.jpg)
Type II Restriction enzymes form blunt or sticky ends.
• EcoRV recognizes 5'-GATATC-3'. • Complimentary sequence is implied. • EcoRV cuts both strands after the 5'-T. • Each fragment has blunt ends.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 50: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/50.jpg)
DNA fragment sizes can be quantified from gel images.
• Gel electrophoresis is a method of separating DNA fragments by size.
• Gel is made of agarose.
• DNA bands can be stained with chemicals such as ethidium bromide for fluorescent detection.
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 51: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/51.jpg)
What is a DNA Plasmid? • Double-stranded, extra-chromosomal DNA • Size: 1–200 kbases • Covalently closed, circular, superhelical • Bacterial • Dependent on host cell’s proteins for replication
and transcription machinery
© 2014 John Wiley & Sons, Inc. All rights reserved.
![Page 52: Essential Biochemistry - StFXWater plays a role in dictating biomolecular structure via the hydrophobic effect. The hydrophobic effect is the phenomenon by which nonpolar molecules](https://reader034.vdocument.in/reader034/viewer/2022042219/5ec4f7bd06d95415bb142b8c/html5/thumbnails/52.jpg)
Site-Directed Mutagenesis
A method of introducing a single amino acid change in a protein as a result of a mutation at a specific site in the DNA.
© 2014 John Wiley & Sons, Inc. All rights reserved.