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GUMEDE ESTHER NTOMBIFUTHI 3RD YEAR
UNIVERSITY OF JOHANNESBURG 2014
NUCLEIC ACIDS :
DNA Structure
DNA consists of two molecules that are

arranged into a ladder-like structure called a
Double Helix.

A mo...
Nucleotides
Phosphate

Nitrogenous
Base
Pentose
Sugar
DNA Structure Helps
Explain How It Duplicates
DNA is made up of two nucleotide

strands held together by hydrogen bonds
...
DEOXYRIBONUCLEIC ACID
DNA usually exists as a double-stranded
structure, with both strands coiled
together to form the cha...
Nucleotides
The phosphate and sugar form the

backbone of the DNA molecule, whereas the
bases form the “rungs”.

There a...
Orientation of
DNA on the sugar ring are
The carbon atoms
numbered for reference. The 5’ and 3’
hydroxyl groups (highlight...
Nucleotides
A

Adenine

C

Cytosine

T

Thymine

G

Guanine
Nucleotides
Each base will only bond with one other

specific base.

Adenine (A)
Thymine (T)
Cytosine (C)
Guanine (G)...
DNA Structure
A gene is a section of DNA that codes for a

protein.

Each unique gene has a unique sequence of

bases.

...
DNA
Gene
Protein

Trait
A Nucleoside is a combination of
Pentose sugar & Nitrogen Base

A Nucleotide is a combination
of nucleoside & Phosphoric A...
P
T

A

5’
C

G

P

3’
P

DNA has directionality.

P
P
P
C

Two nucleotide chains
together wind into a helix.

G
A

P

P

...
DNA BACKBONE

03/07/14

Pranabjyoti Das

15
Nucleotides are matched between strands
through hydrogen bonds to form base pairs.
Adenine pairs with thymine and cytosine...
These terms refer to the
carbon atom in
deoxyribose to which the
next phosphate in the
chain attaches.
Directionality has
...
The pairing of bases in DNA
through hydrogen bonding
means that the information
contained within each strand is
redundant....
Functions
DNA is used to store genetic information
It is replicated before cell division

DNA is very important so it i...
03/07/14

Pranabjyoti Das

20
DNA replication is a biological
process that occurs in all living
organisms and copies their exact
DNA. It is the basis fo...
 Each old strand
stays intact
 Each new DNA
molecule is half
“old” and half
“new”

Fig. 1-7, p.212
The first major step for the DNA
Replication to take place is the
breaking of hydrogen bonds between
bases of the two anti...
Helicase is the enzyme that
splits the two strands. The
structure that is created is
known as "Replication
Fork".

03/07/1...
03/07/14

In order for DNA replication to begin,
the double stranded DNA helix must first
be opened. The sites where this ...
Replication

3’
3’

5’

5’
3’
5’

3’

5’

Helicase protein binds to DNA sequences called
origins and unwinds DNA strands.
...
Overall direction
of replication

3’
3’

5’

5’
3’
5’

3’
5’

DNA polymerase enzyme adds DNA nucleotides
to the RNA primer...
Overall direction
of replication

3’
3’

5’

5’
3’
5’

Leading strand synthesis continues in a
5’ to 3’ direction.

3’
5’
Overall direction
of replication

3’
3’

5’

5’

Okazaki fragment

3’
5’

3’ 5’

Leading strand synthesis continues in a
5...
rk
o

F
o n The replication fork is a structure that
ti
forms within the nucleus during DNA
ca
i
pl
replication. It is cre...
3’
5’

3’
5’
3’
5’

3’
5’

DNA polymerase enzyme adds DNA nucleotides
to the RNA primer.
DNA polymerase proofreads bases a...
3’
5’

3’
5’
3’
5’

3’5’

3’5’

3’
5’

Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesis...
3’
3’

5’

5’

Okazaki fragment

3’
5’

3’ 5’

Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous s...
3’
5’

3’
5’
3’
5’

3’ 5’

3’5’

3’
5’

Leading strand synthesis continues in a
5’ to 3’ direction.
Discontinuous synthesi...
5’

3’

3’
5’
3’

3’5’
5’

3’5’

3’
5’

Exonuclease enzymes remove RNA primers.
3’
3’
5’
3’
5’

3’5’

3’
5’

Exonuclease enzymes remove RNA primers.
Ligase forms bonds between sugar-phosphate
backbone.
One of the most important steps of DNA
Replication is
the
binding
of RNA
Primase in the initiation point of the 3'-5'
pare...
In the lagging strand the DNA Pol IExonuclease- reads the fragments and
removes the RNA Primers. The gaps are
closed with ...
Enzymes in Replication
Enzymes (Helicases) unwind the two

strands
DNA polymerase needed for the synthesis of

complemen...
Helicase unwinds
parental double helix

DNA polymerase
binds nucleotides
to form new strands

Binding proteins
stabilize s...
DNA Replication models
There are three possible models that

describe the accurate creation of the
daughter chains:

Sem...
Figure 11.2

11-6
Replicatoin can’t just start:
1. All DNA polymerases need a primer
2. The primer can be a piece of RNA or DNA
3. It must b...
“Proof-reading” is essential at DNA replication

•What is proof-reading?
1. If there is a wrong base built in, then there ...
Klenow Fragment (of pol I)
(The proof-reading activity)
 Bacterial DNA polymerases may vary in their
subunit composition


However, they have the same type of catalytic subunit...
Proteins involved in E. coli replication

Terms to be known
DNA replication mistakes
The most errors in DNA sequence occur during

replication.
Reparation takes place after replica...
Is a method in which multiple repetitions of DNA
replication are performed in a test tube.
Mix in test tube:
DNA template
...
DNA template is denatured with heat to separate strands.

5’

3’

G
C
5’

A
T

A
T

C
G

T
A

A
T

G
C

C
G

G
C
3’
DNA template is denatured with heat to separate strands.
5’

3’

G

A

C

T

A

G

C

G

C
5’

A

T

T

G

A

T

C

G

C
3...
Each DNA primer anneals, binding to its
complementary sequence on the template DNA
5’

3’

G
C

A
T

A
T

5’

C

T

A

G

...
DNA polymerase creates a new strand of DNA
complementary to the template DNA starting from the
primer.
3’

G
C

A
T

A
T

...
Denaturation

DNA template is denatured with
high heat to separate strands.

Annealing

Each DNA primer anneals, binding
t...
Visualization of PCR products
agarose gel-electrophoresis of DNA
Top
Negative pole
DNA is
Negatively charged
Small molecules
dissolve faster

separation...
Dna
Dna
Dna
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  • Transcript of "Dna"

    1. 1. GUMEDE ESTHER NTOMBIFUTHI 3RD YEAR UNIVERSITY OF JOHANNESBURG 2014
    2. 2. NUCLEIC ACIDS :
    3. 3. DNA Structure DNA consists of two molecules that are arranged into a ladder-like structure called a Double Helix. A molecule of DNA is made up of millions of tiny subunits called Nucleotides. Each nucleotide consists of: 1. Phosphate group 2. Pentose sugar-Deoxyribose 3. Nitrogenous base
    4. 4. Nucleotides Phosphate Nitrogenous Base Pentose Sugar
    5. 5. DNA Structure Helps Explain How It Duplicates DNA is made up of two nucleotide strands held together by hydrogen bonds Hydrogen bonds between two strands are easily broken Each single strand then serves as template for new strand
    6. 6. DEOXYRIBONUCLEIC ACID DNA usually exists as a double-stranded structure, with both strands coiled together to form the characteristic double-helix. Each single strand of DNA is a chain of four types of nucleotides having the bases: Adenine Cytosine Guanine Thymine 03/07/14 Pranabjyoti Das 6
    7. 7. Nucleotides The phosphate and sugar form the backbone of the DNA molecule, whereas the bases form the “rungs”. There are four types of nitrogenous bases.
    8. 8. Orientation of DNA on the sugar ring are The carbon atoms numbered for reference. The 5’ and 3’ hydroxyl groups (highlighted on the left) are used to attach phosphate groups.  The directionality of a DNA strand is due to the orientation of the phosphate-sugar backbone.
    9. 9. Nucleotides A Adenine C Cytosine T Thymine G Guanine
    10. 10. Nucleotides Each base will only bond with one other specific base. Adenine (A) Thymine (T) Cytosine (C) Guanine (G) Form a base pair. Form a base pair.
    11. 11. DNA Structure A gene is a section of DNA that codes for a protein. Each unique gene has a unique sequence of bases. This unique sequence of bases will code for the production of a unique protein. It is these proteins and combination of proteins that give us a unique phenotype.
    12. 12. DNA Gene Protein Trait
    13. 13. A Nucleoside is a combination of Pentose sugar & Nitrogen Base A Nucleotide is a combination of nucleoside & Phosphoric Acid 03/07/14 Pranabjyoti Das 13
    14. 14. P T A 5’ C G P 3’ P DNA has directionality. P P P C Two nucleotide chains together wind into a helix. G A P P T P P G Hydrogen bonds between paired bases hold the two DNA strands together. C P P G 3’ C DNA strands are antiparallel. P P A sugar and phosphate “backbone” connects nucleotides in a chain. 5’
    15. 15. DNA BACKBONE 03/07/14 Pranabjyoti Das 15
    16. 16. Nucleotides are matched between strands through hydrogen bonds to form base pairs. Adenine pairs with thymine and cytosine pairs with guanine 03/07/14 Pranabjyoti Das 16
    17. 17. These terms refer to the carbon atom in deoxyribose to which the next phosphate in the chain attaches. Directionality has consequences in DNA synthesis, because DNA polymerase can synthesize DNA in only one direction by adding nucleotides to the 3' end of a DNA strand. 03/07/14 Pranabjyoti Das 17
    18. 18. The pairing of bases in DNA through hydrogen bonding means that the information contained within each strand is redundant. The nucleotides on a single strand can be used to reconstruct nucleotides on a newly synthesized partner strand. 03/07/14 Pranabjyoti Das 18
    19. 19. Functions DNA is used to store genetic information It is replicated before cell division DNA is very important so it is stored in the nucleus. It never leaves the nucleus Your DNA stores the code for your proteins, which exhibit your “traits” The DNA gets converted to RNA in order to move out into the cytoplasm
    20. 20. 03/07/14 Pranabjyoti Das 20
    21. 21. DNA replication is a biological process that occurs in all living organisms and copies their exact DNA. It is the basis for biological inheritance. 03/07/14 Pranabjyoti Das 21
    22. 22.  Each old strand stays intact  Each new DNA molecule is half “old” and half “new” Fig. 1-7, p.212
    23. 23. The first major step for the DNA Replication to take place is the breaking of hydrogen bonds between bases of the two antiparallel strands. The unwounding of the two strands is the starting point. The splitting happens in places of the chains which are rich in A-T. That is because there are only two bonds between Adenine and Thymine. There are three hydrogen bonds between Cytosine and Guanine.  03/07/14 Pranabjyoti Das 23
    24. 24. Helicase is the enzyme that splits the two strands. The structure that is created is known as "Replication Fork". 03/07/14 Pranabjyoti Das 25
    25. 25. 03/07/14 In order for DNA replication to begin, the double stranded DNA helix must first be opened. The sites where this process first occurs are called replication origins. Helicase unwinds the two single strands 26 Pranabjyoti Das
    26. 26. Replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ Helicase protein binds to DNA sequences called origins and unwinds DNA strands. Binding proteins prevent single strands from rewinding. Primase protein makes a short segment of RNA complementary to the DNA, a primer.
    27. 27. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ 3’ 5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer.
    28. 28. Overall direction of replication 3’ 3’ 5’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. 3’ 5’
    29. 29. Overall direction of replication 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. 3’ 5’
    30. 30. rk o F o n The replication fork is a structure that ti forms within the nucleus during DNA ca i pl replication. It is created by helicases, e R which break the hydrogen bonds holding the two DNA strands together. The resulting structure has two branching "prongs", each one made up of a single strand of DNA. These two strands serve as the template for the leading and lagging strands, which will be created as DNA polymerase matches complementary nucleotides to the templates; The templates may be properly referred to as the leading strand template and the lagging strand template 03/07/14 Pranabjyoti Das 31
    31. 31. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ DNA polymerase enzyme adds DNA nucleotides to the RNA primer. DNA polymerase proofreads bases added and replaces incorrect nucleotides.
    32. 32. 3’ 5’ 3’ 5’ 3’ 5’ 3’5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
    33. 33. 3’ 3’ 5’ 5’ Okazaki fragment 3’ 5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments. 3’ 5’
    34. 34. 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ Leading strand synthesis continues in a 5’ to 3’ direction. Discontinuous synthesis produces 5’ to 3’ DNA segments called Okazaki fragments.
    35. 35. 5’ 3’ 3’ 5’ 3’ 3’5’ 5’ 3’5’ 3’ 5’ Exonuclease enzymes remove RNA primers.
    36. 36. 3’ 3’ 5’ 3’ 5’ 3’5’ 3’ 5’ Exonuclease enzymes remove RNA primers. Ligase forms bonds between sugar-phosphate backbone.
    37. 37. One of the most important steps of DNA Replication is the binding of RNA Primase in the initiation point of the 3'-5' parent chain.  RNA Primase can attract RNA nucleotides which bind to the DNA nucleotides of the 3'-5' strand due to the hydrogen bonds between the bases. RNA nucleotides are the primers (starters) for the binding of DNA nucleotides.  03/07/14 Pranabjyoti Das 38
    38. 38. In the lagging strand the DNA Pol IExonuclease- reads the fragments and removes the RNA Primers. The gaps are closed with the action of DNA Polymerase which adds complementary nucleotides to the gaps and DNA Ligase which acts as a glue to attach the phosphate to the sugar by forming phosphodiester bond. 03/07/14 Pranabjyoti Das 39
    39. 39. Enzymes in Replication Enzymes (Helicases) unwind the two strands DNA polymerase needed for the synthesis of complementary strand DNA ligase joins pieces of the lagging strand together
    40. 40. Helicase unwinds parental double helix DNA polymerase binds nucleotides to form new strands Binding proteins stabilize separate strands Exonuclease removes RNA primer and inserts the correct bases Primase adds short primer to template strand Ligase joins Okazaki fragments and seals other nicks in sugarphosphate backbone
    41. 41. DNA Replication models There are three possible models that describe the accurate creation of the daughter chains: Semiconservative Replication Conservative Replication Dispersive Replication
    42. 42. Figure 11.2 11-6
    43. 43. Replicatoin can’t just start: 1. All DNA polymerases need a primer 2. The primer can be a piece of RNA or DNA 3. It must be “base-paired” with the “template” and with primer 3’OH Thus: 5’ 3’ 3’ Synthesis direction 5’ Template strand
    44. 44. “Proof-reading” is essential at DNA replication •What is proof-reading? 1. If there is a wrong base built in, then there is no base paring possible. 2. The DNA polymerase can’t continue on building in the next base. 3. The DNA polymerase removes the “wrong” base and starts over
    45. 45. Klenow Fragment (of pol I) (The proof-reading activity)
    46. 46.  Bacterial DNA polymerases may vary in their subunit composition  However, they have the same type of catalytic subunit Structure resembles a human right hand Template DNA thread through the palm; Thumb and fingers wrapped around the DNA
    47. 47. Proteins involved in E. coli replication Terms to be known
    48. 48. DNA replication mistakes The most errors in DNA sequence occur during replication. Reparation takes place after replication is finished DNA polymerases can get the right sequence from the complementary strand and repair, along with DNA ligase, the wrong bases.
    49. 49. Is a method in which multiple repetitions of DNA replication are performed in a test tube. Mix in test tube: DNA template DNA to be amplified Primers one complementary to each strand Nucleotides dATP,d GTP, dCTP, and dTTP DNA polymerase heat stable form from thermophilic bacteria
    50. 50. DNA template is denatured with heat to separate strands. 5’ 3’ G C 5’ A T A T C G T A A T G C C G G C 3’
    51. 51. DNA template is denatured with heat to separate strands. 5’ 3’ G A C T A G C G C 5’ A T T G A T C G C 3’
    52. 52. Each DNA primer anneals, binding to its complementary sequence on the template DNA 5’ 3’ G C A T A T 5’ C T A G C G 3’ 5’ 3’ C 5’ T T G A T G C C G G C 3’
    53. 53. DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. 3’ G C A T A T C G T A A T G C C G G C 3’ 5’ 5’ 3’ G C 5’ 5’ A T A T C G T A A T G C C G G C 3’
    54. 54. Denaturation DNA template is denatured with high heat to separate strands. Annealing Each DNA primer anneals, binding to its complementary sequence on the template DNA Extension DNA polymerase creates a new strand of DNA complementary to the template DNA starting from the primer. Multiple rounds of denaturation-annealing-extension are performed to create many copies of the template DNA between the two primer sequences.
    55. 55. Visualization of PCR products
    56. 56. agarose gel-electrophoresis of DNA Top Negative pole DNA is Negatively charged Small molecules dissolve faster separation based on size Bottom Positive pole Slab of agarose gel (ethidium bromide staining)
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