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FOR
ANY
EXAM
PREPARATION
&
BASIC GUIDENCES
OF
MOLECULAR
BIOLOGY &
GENETICS
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PREFACE
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followed by Molecular Biology. It provides students with
genetic matrieal background to enable them to
understand and critically analyze recent advances in
laboratory sciences.
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`
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TABLE OF CONTENTS
Preface
Acknowledgement
Table of Contents
List of Figures
General objectives
3.0. Introduction
3.1. Mapping
3.2. Double Crossovers
3.3. Interference
3.4. Deriving Linkage Distance and Gene Order from
Three-Point Crosses
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CHAPTER 4 : PEDIGREE ANALYSIS (76-91)
5.0. Introduction
5.1. Deoxyribonucleic acid
5.2. Ribonucleic acid
5.3. Chemical differences between DNA & RNA
5.4. DNA Replication
5.5. Control of Replication
5.6. DNA Ligation
6.0. Introduction
6.1. Agents that Damage DNA
6.2. Types of DNA damage
6.3. Repairing Damaged Bases
6.4. Repairing Strand Breaks
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6.5. Mutation
6.6. Insertions and Deletions
6.7. Duplications
6.8. Translocations
6.9. Frequency of Mutations
6.10.Measuring Mutation Rate
7.0. Introduction
7.1. Conjugation
7.2. Transduction
7.3. Transformation
7.4. Transposition
7.5. Recombination
7.6. Plasmid
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CHAPTER 9 : CONTROL OF GENE EXPRESSION (200-233)
9.0. Introduction
9.1. Gene Control in Prokaryotes
9.2. The lac Operon
9.2. The trp Operon
9.3. Gene Control in Eukaryotes
10.0. Introduction
10.1. Uses of Genetic Engineering
10.2. Basic Tools of Genetic Engineering
10.3. Enzymes in Molecular Biology
10.4. DNA manipulation
10.5. Making a Recombinant DNA: An Overview
10.6. Cloning
10.7. Cloning DNA
10.8. Cloning into a Plasmid
10.9. Expression and Engineering of Macromolecules
10.10. Creating mutations
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CHAPTER 11: DNA SEQUENCING (287-311)
11.0. Introduction
11.1. Sanger Method for DNA Sequencing
11.2. An Automated sequencing gel
11.3. Shotgun Sequencing
12. 1. Electrophoresis
12.2. Complementarity and Hybridization
12.3. Blots
12.4. Polymerase Chain Reaction
12.5. RFLP
12.6. DNA Finger printing
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List of Figures
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Fig.16. Steps in breaking the genetic code: the deciphering of a
poly-U mRNA ................................................................. 182
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INTRODUCTION
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The essential characteristic of Molecular Genetics is that
gene products are studied through the genes that
encode them. This contrasts with a biochemical
approach, in which the gene products themselves are
purified and their activities studied in vitro.
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gene/protein does, the resulting conclusions are much
stronger than if one only use one of these strategies.
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1966 -Gene transcription become reality
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CHAPTER 1
GENETICS
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yellow or green
4. seed shape is
round or wrinkled
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Lethal alleles
Many so called dominant mutations are in fact
semidominant, the phenotype of the homozygote is
more extreme than the phenotype of the
heterozygote. For instance the gene T (Danforth's
short tail) in mice. The normal allele of this gene is
expressed in the embryo. T/+ mice develop a short
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T/+ x T/+
|
|
V
T/T T/+ +/+
1 : 2 : 1 ratio at conception
0 : 2 : 1 ratio at birth
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Codominance
If two or more alleles can each be distinguished in
the phenotype in the presence of the other they are
said to be codominant. An example is seen in the
ABO blood group where the A and B alleles are
codominant.
The ABO gene codes for a glycosyl-transferase
which modifies the H antigen on the surface of red
blood cells. The A form adds N-acetylgalactosamine,
the B form adds D-galactose forming the A and B
antigens respectively. The O allele has a frameshift
mutation in the gene and thus produces a truncated
and inactive product which cannot modify H. A
phenotype people have natural antibodies to B
antigen in their serum and vice versa. O phenotype
individuals have antibodies directed against both A
and B. AB individuals have no antibodies against
either A or B antigens.
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AA A A anti-B
AO A A anti-B
BB B B anti-A
BO B B anti-A
AB AB A and B neither
anti-A and
OO O neither
anti-B
Silent alleles
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Epistasis
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Pleiotropy
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Genetic heterogeneity
This is the term used to describe a condition which may
be caused by mutations in more than one gene.
Tuberous sclerosis again provides a good example of
this, the identical disease is produced by mutations in
either of two unrelated genes, TSC1 on chromosome 9
or TSC2 on chromosome 16. In such cases, presumably
both genes act at different points in the same
biochemical or regulatory pathway. Or perhaps one
provides a ligand and one a receptor.
variable expressivity
The degree to which a disease may manifest itself can
be very variable and, once again, tuberous sclerosis
provides a good example. Some individuals scarcely
have any symptoms at all whereas others are severely
affected. Sometimes very mild symptoms may be
overlooked and then a person may be wrongly classified
as non-affected. Clearly this could have profound
implications for genetic counselling.
Incomplete Penetrance
This is an extreme case of a low level of expressivity
Some individuals who logically ought to show symptoms
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Anticipation
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5 normal
19 - 30 "pre-mutant"
myotonic dystrophy
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Germline Mosaicism
Phenocopies
mitochondrial inheritance
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Uniparental disomy
Although it is not possible to make a viable human
embryo with two complete haploid sets of chromosomes
from the same sex parent it is sometimes possible that
both copies of a single chromosome may be inherited
from the same parent (along with no copies of the
corresponding chromosome from the other parent.) Rare
cases of cystic fibrosis (a common autosomal recessive
disease) have occurred in which one parent was a
heterozygous carrier of the disease but the second
parent had two wild type alleles. The child had received
two copies of the mutant chromosome 7 from the carrier
parent and no chromosome 7 from the unaffected
parent.
linkage
When two genes are close together on the same
chromosome they tend to be inherited together because
of the mechanics of chromosome segregation at
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Review Questions
1. Why Mendel uses Pea plants for his experiment?
Co-dominance
Allele
Gene
Recessive
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CHAPTER 2
CHROMOSOME STRUCTURE AND
FUNCTION
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Heterochromatin
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Euchromatin
Euchromatin is threadlike, delicate.
It is most abundant in active, transcribing cells.
Thus, the presence of euchromatin is significant
because the regions of DNA to be transcribed or
duplicated must uncoil before the genetic code can
be read.
Euchromatin stains weakly and is more open (less
condensed).
Euchromatin remains dispersed (uncondensed)
during Interphase, when RNA transcription occurs.
As the cell differentiates, the proportion of
heterochromatin to euchromatin increases, reflecting
increased specialization of the cell as it matures.
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2.8. Epistasis
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1. Height
2. Systemic Lupus Erythematus
3. Weight
4. Eye Color
5. Intelligence
6. Skin Color
7. Many forms of behavior
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2.11. Pleiotropy
2) Cats that are white with blue eyes are often deaf,
white cats with a blue and an yellow-orange eye
are deaf on the side with the blue eye.
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118
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2.13. Cytogenetics
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Review Questions
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CHAPTER 3
LINKAGE
Specific learning objectives
At the end of this chapter, students are expected
to
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3.0. Introduction
Linkage occurs when genes are on the same
chromosome. When genes occur on the same
chromosome, they are inherited as a single unit.
Genes inherited in this way are called Linked.
Genes are located on specific regions of a certain
chromosome, termed the gene locus (plural: loci). A
gene therefore is a specific segment of the DNA
molecule.
Linkage groups are invariably the same number as
the pairs of homologous chromosomes an organism
possesses. Recombination occurs when crossing-
over has broken linkage groups, as in the case of
the genes for wing size and body color that Morgan
studied.
Chromosome mapping was originally based on the
frequencies of recombination between alleles.
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Frizzled 18 63 81
Normal 63 13 76
81 76 157
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White Coloured
Frizzled 15 2 17
Normal 4 12 16
19 14 33
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3.1. Mapping
If one can arrange testcrosses for triple (or higher order)
heterozygotes and recessives (a three-point cross), the
recombination can be calculated for the three pairs of
genes. The data will look like this example:
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1 ABC 17959
2 abc 17699
3 Abc 509
4 aBC 524
5 ABc 4455
6 abC 4654
7 AbC 20
8 aBc 12
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A a
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Locus Coord
csu95a 0.00
csh2c(cdc2) 144.60
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3.3. Interference
The term interference refers to the fact that
recombination seems to be suppressed close to a first
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I = 1-c
c = Coefficent of coincidence
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v cv+ ct 3 Double-crossover
v+ cv ct+ 5 Double-crossover
Total 1448
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Review Questions
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Phenotype # Observed
Wild Type 405
Normal, vestigial 85
Black, normal 100
Black, vestigial 410
Are these two genes linked? How did you come to this
conclusion? What calculation would you perform to
confirm you conclusion?
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CHAPTER 4
PEDIGREE ANALYSIS
Specific learning objectives
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4.1. Introduction
A pedigree is a diagram of family relationships that uses
symbols to represent people and lines to represent
genetic relationships. These diagrams make it easier to
visualize relationships within families, particularly large
extended families. Pedigrees are often used to
determine the mode of inheritance (dominant, recessive,
etc.) of genetic diseases.
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Non-inheritedtraits
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- Tuberous sclerosis,
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Review Questions
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CHAPTER 5
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5.0. Introduction
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The two strands coil about each other so that all the
bases project inward towards the helix axis. The two
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In prokaryotes
In eukaryotic cells
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The Steps:
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LINEAR GENOMES
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OTHER GENOMES
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Early replication
Bacteriophage lambda initially replicates by means of
theta form intermediates. The origin of replication (ori)
is located within the O gene, whose product is required
for replication. The gene P product is also required for
replication.
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Late replication
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Replication of Bacteriophage T7
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o MCM proteins. Only DNA coated with MCM
proteins (there are 6 of them) can be
replicated.
Once replication begins in S phase,
o CDC-6 and CDT-1 leave the ORCs (the
latter by ubiquination and destruction in
proteasomes).
o The MCM proteins leave in front of the
advancing replication fork.
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In Summary,
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Review Questions
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CHAPTER 6
6.0. Introduction
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6.5. Mutations
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6. 7. Duplications
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6.8. Translocations
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Autosomal dominants
o Retinoblastoma in the RB gene [Link]:
about 8 per million (8 x 10-6)
o Osteogenesis imperfecta in one or the
other of the two genes that encode Type I
collagen: about 1 per 100,000 (10-5)
o Inherited tendency to polyps (and later
cancer) in the colon. in a tumor
suppressor gene (APC)~10-5
X-linked recessives
o Hemophilia A ~3 x 10-5 (the Factor VIII
gene)
o Duchenne Muscular Dystrophy (DMD) >8
x 10-5 (the dystrophin gene) Why should
the mutation frequency in the dystrophin
gene be so much larger than most of the
others? It's probably a matter of size. The
dystrophin gene stretches over 2.3 x 106
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Review Questions
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CHAPTER 7
GENE TRANSFER IN BACTERIA
7.0. Introdction
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7.1. Conjugation
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Hfr conjugation
Donors: Hfr bacteria perform as donors during
conjugation. One strand of the chromosome
copy is transferred to the recipient F- cells,
while the other strand remains in the Hfr cell.
The donor remains unchanged genetically.
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Transfer
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F F
Conjugation
F+ F-
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F′ Factors
- F factor plasmid with some chromosomal
material, which occur as a result of imprecise
exicision, is called an F1 factor.
- An F' factor can conjugate. The recipient
becomes F', and information passed across is
part chromosome and part plasmid.
7.2. Transformation
Griffith's Experiment
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Process of transfer
1. Revessible association of DNA to the cell wall is
mediated by an ionic interaction between DNA and
the cell wall of competent organism.
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7.3. Transduction
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7.4.Transposons
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7.5. Recombination
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7.6. Plasmids
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Reviwe Questions
1. Compare and contrast the following terms
- Conjugation
- Transformation
- Transduction
- Transposition
- Recombination
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CHAPTER 8
TRANSCRIPTION AND
TRANSLATION
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8.0. Introduction
The Steps
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8.1 Transcription
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Fig.16 Transcription
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8.2. Translation
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3’-TAGCATGAT-5’?
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o 5'-AGTCAATGCAAGTTCCATGCAT....
A gene determine the sequence of amino acids
in proteins. We use a shorthand notation to write
a protein sequence:
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Fig.21 A polysome
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Review Questions
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CHAPTER 9
CONTROL OF GENE EXPRESSION
9.0. Introduction
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The trp operon (see Fig 10.2. below) encodes the genes
for the synthesis of tryptophan. This cluster of genes,
like the lac operon, is regulated by a repressor that
binds to the operator sequences. The activity of the trp
repressor for binding the operator region is enhanced
when it binds tryptophan; in this capacity, tryptophan is
known as a corepressor. Since the activity of the trp
repressor is enhanced in the presence of tryptophan, the
rate of expression of the trp operon is graded in
response to the level of tryptophan in the cell.
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First exon
Intron bordered by a splice donor and splice
acceptor and containing a splice branchpoint
3' UTR
polyA+ tail
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ORFs)
no splicing
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Review Questions
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CHAPTER 10
RECOMBINANT DNA
TECHNOLOGY
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10.0. Introduction
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are various things that one can do with the DNA that has
been transferred to a recipient cell. Note that the
transferred DNA may be from the same species or from
a different species than the recipient. Such successfully
transferred DNA is said to be cloned.
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I. Nulease
There are two types of nucleases.
A. Exonucleases -
- Cleaves nucleotide one at a time from the end of
a nucleic acid
B. Endonucleases
- Cleaved bonds within a contiguous molecule of
nucleic acid.
1. Restriction enzymes
- Endonucleases:- cleave dsDNA,
- do not cleave ssDNA or RNA
- Each recognizes a specific nucleotide sequence,
often palindromic.
Palindromic is a state where both strands have the
same nucleotide sequence but in anti-parallel directions
1. 6- cutters - recognize 6 nucleotide sequences
- less frequent sites within DNA, in 4100bp
e.g. Bamtel Gl GATCC
EcoRI GlAA TTC
2. 4- cutters - recognize 4 nucleotide sequences
- More frequent, in 256bp
e.g- Sau3AGATC,
HaelllGGCC
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II. RNases
- Endonucleases cleave at specific ribonucleotides
A. RNAse A cleaves ssRNA at pyrimidine
nucleotides (C,U)
51 A-G-Cl-G-A-Ul-GC-ACl-A3 1 + RNase A
A-G-C G-A-U G-C A-C A
B. RNase T1 cleaves ssRNA at G
nucleotides 5 A-Gl-C-G-A-U-Gl-A-C-A3
lRNAse Tl AG C-G AUG A-C-A
b. 51 → 31 exanuclease
c. 31 → 51
exanuclease
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2.2. Klenow
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a. 51 → 31 DNA polymerase
b. exoribonvclease 9specifically degrades RNA in a
DNA: RNA hybrid.
- It is used to transcribe first cDNA in cDNA
cloning.
IV.RNA polymerases
- They are isolated from various
bacteriophages including
a. Phage SP6
b. Phage T7
c. Phage T3
- Its activity is to synthesize ssRNA from a
DNA template
- Its uses include to synthesize sequence
specific RNA probes which is used for:
a. Labeling 51 ends of synthetic
oligonuceoties
b. Phosphorylating synthetic linkers and
other synthetic DNA fragments lacking a
31- prior to ligation
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V. T4 polynucleotide kinase
- It is obtained from T4 plasmid infected E.coli
- It activities include:
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1. Nick translation
2. In vitro transcription
3. cDNA synthesis
4. End labeling
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The ends of the cut have an overhanging piece
of single-stranded DNA.
These are called "sticky ends" because they are
able to base pair with any DNA molecule
containing the complementary sticky end.
In this case, both DNA preparations have
complementary sticky ends and thus can pair
with each other when mixed.
DNA ligase covalently links the two into a
molecule of recombinant DNA.
10.6. Cloning
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The key is that the EcoRI site is within the kanr gene, so
when a piece of human DNA is inserted there, the
gene's function is destroyed.
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So,
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Features
Relatively small, 4.36kb. This size provides easy
purification from E.coli cells
Relaxed origin of replication gives high copy
number of plasmids within each bacterium (200-
1000 plasmid copies)
Tow antibiotic resistance genes, (AMP) and
Tetracycline (Tet)
Unique pst1 site within the AMpiclin gene and
unique BamHI side within the tetracycline gene.
This unique restriction target sites are
useful in cloning
2. Phage - derivatives of bacteriophage lambda; linear
DNA molecules, whose region can be replaced with
foreign DNA without disrupting its life cycle;
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2.1.λ-phage
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Procedures:
1. Isolating DNA
2. Cutting DNA
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3. Joining DNA
Donor DNA and vector DNA are digested with the same
restriction enzyme and mixed in a test tube in order to
allow the ends to join to each other and form
recombinant DNA. At this stage the sugar-phosphate
backbones are still not complete at two positions at each
junction.
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I. Expression
DNA Synthesis
Automated DNA synthesis machines allow one
to obtain single stranded molecules of any
specific or randomized sequence up to 100
nucleotides long. Cycle times are a few minutes
per base.
RNA In vitro transcription
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Expression in Ecoli
Up to 50% of total soluble protein can often be
obtained. Possible problems include solubility
(formation of inclusion bodies), lethality
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II. Manipulation
III. Selections
The ability to generate large populations of molecules of
different sequence coupled with a method for linking
their replication to their ability to function creates new
opportunities to identify functional molecules that may
never have arisen in nature. Comparing these novel
macromolecules to each other and to their natural
counterparts may enable us to overcome the limitations
imposed by the patchy, historical sampling of possible
sequences which occurred during evolution and achieve
new insights on the rules underlying the function of
proteins and nucleic acids.
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D. Cassette mutagenesis
A double strand synthetic DNA cassette with
ends complimentary to existing restriction sites is
required.
E. Combinatorial mutagenesis
With either technology, mixtures of DNA bases can
replace pure solutions at selected positions in the
sequence to be synthesized. The result is a
population of individual molecules, each with a
different sequence. Upon transformation of E. coli
individual DNA molecules give rise to pure clones. In
this way many different variants can be obtained in a
single experiment. If the randomization was such
that small number of variants are expected
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Review Questions
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9. What is a cDNA?
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CHAPTER 11
DNA SEQUENCING
11.0. Introduction
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a. DNA segment
c. DNA polymerase
d. Primer
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3’ ATGGACTGCAT 5’ watson
5’ TA-stop
3’ ATGGACTGCAT 5’ watson
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5’ TACCTG-stop
3’ ATGGACTGCAT 5’ watson
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The dideoxy method gets its name from the critical role
played by synthetic nucleotides that lack the -OH at the
3′ carbon atom (red arrow). A dideoxynucleotide
(dideoxythymidine triphosphate — ddTTP — is the one
shown here) can be added to the growing DNA strand
but when it is, chain elongation stops because there is
no 3′ -OH for the next nucleotide to be attached to. For
this reason, the dideoxy method is also called the chain
termination method.
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11.1.1. Procedure
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that only the bands with the radioactive label on the 5'
end will appear. In PAGE, the shortest fragments will
migrate the farthest. Therefore, the bottom-most band
indicates that its particular dideoxynucleotide was added
first to the labeled primer.
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Review Questions
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CHAPTER 12
MOLECULAR TECHNIQUES
12.1. Electrophoresis
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12.3. Blots
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• Gel electrophoresis
• Transfer to Solid Support
• Blocking
• Preparing the Probe
• Hybridization
• Washing
• Detection of Probe-Target Hybrids
12.3.1. Gel Electrophoresis
This is a technique that separates molecules on the
basis of their size. First, a slab of gel material is cast.
Gels are usually cast from agarose or poly-acrylamide.
These gels are solid and consist of a matrix of long thin
molecules forming sub-microscopic pores. The size of
the pores can be controlled by varying the chemical
composition of the gel. The gel is cast soaked with
buffer.
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Staining DNA
DNA is stained with ethidium bromide (EtBr),
which binds to nucleic aids. The DNA-EtBr
complex fluoresces under UV light.
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Staining RNA
RNA is stained with ethidium bromide (EtBr),
which binds to nucleic aids. The RNA-EtBr
complex fluoresces under UV light.
Staining Protein
Protein is stained with Coomassie Blue (CB).
The protein-CB complex is deep blue and can be
seen with visible light.
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RNA and protein are run in the gels in a state that allows
the probe to bind without this pre-treatment.
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12.3.3. Blocking
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12.3.5. Hybridization
12.3.6. Washing
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12.3.7.1. Autoradiography
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Introduction
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o Molecular cloning o
Pathogen detection o
Genetic engineering o
Mutagenesis
o Genetics, producing molecular markers o
Forensics
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cycler, which can heat and cool the tubes with the
reaction mixture in a very short time.
1. Denaturation at 94°C :
2. Annealing at 54°C :
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Once there are a few bases built in, the ionic bond is so
strong between the template and the primer, that it does
not break anymore.
3. Extension at 72°C:
1.02 x 109).
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Fidelity
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Quantification:
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So, the greater the distance between the RFLP and the
gene locus, the lower the probability of an accurate
diagnosis.
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Biological Evidence
FBI and police labs around the U.S. have begun to use
DNA fingerprints to link suspects to biological evidence -
blood or semen stains, hair, or items of clothing - found
at the scene of a crime. Since 1987, hundreds of cases
have been decided with the assistance of DNA
fingerprint evidence.
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Personal Identification
Because every organ or tissue of an individual contains
the same DNA fingerprint, the U.S. armed services have
just begun a program to collect DNA fingerprints from all
personnel for use later, in case they are needed to
identify casualties or persons missing in action. The
DNA method will be far superior to the dogtags, dental
records, and blood typing strategies currently in use.
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Review Questions
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GLOSSARY OF TERMS
COMMONLY USED IN MOLECULAR
BIOLOGY
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bp - "base pair"
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ds - "double-stranded"
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kb - 'kilobase"
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POST-TRANSLATIONAL MODIFICATION -
Modifications made to a polypeptide
molecule after its initial synthesis, this
includes proteolytic cleavages,
phosphorylation, glycosylation, carboxylation,
addition of fatty acid moieties, etc.
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486
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GCCAGCCCCCUAGTGGG... Nucleotide
sequence of mRNA
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chloramphenicol acetyltransderase,
downstream of a gene, promoter, or
translational control element of interest, to
more easily identify successful introduction of
the gene into a host and to measure
transcription and/or translation.
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ss - Single stranded.
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5'-->3'
NNNG AATTCNNN EcoRI cut, 5' overhang
NNNCTTAA GNNN
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3'<--5'
5'-->3'
XXXAGCGC TNNN HaeII cut, 3' overhang
XXXT CGCGANNN 3'<--5'
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TRANSCRIPTION/TRANSLATION REACTION - An in
vitro series of reactions, involving the
synthesis (transcription) of an mRNA from a
plasmid (usually with T7 or SP6 RNA
polymerase), followed by use of the mRNA to
program translation in a cell-free system such
as a rabbit reticulocyte lysate. The
polypeptide product of translation in usually
labelled with [35S]-methionine, and examined
in an SDS-PAGE gel with or without prior
immunoprecipitation. This series of reactions
permits the synthesis of a polypeptide from
DNA in vitro.
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species.
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