Applied genetics Questions وراثة تطبيقية
Applied genetics Questions1.
DNA sequencing has become a powerful technique in molecular biology
this
tool has been applied to many areas of research . write in this point and
Shed light on DNA sequencing application in molecular biology ??
1.
the
polymerase chain reaction(PCR) requires first knowing the flanking
sequence of this piece.
2.
amino
acid sequences can be determined more easily by sequencing a piece of cDNA and
finding an open reading frame.
3.
In
eukaryotic gene expression, identify conserved sequence motifs and determine
their importance in the promoter region.
4.
identifying
restriction sites in plasmids. Knowing these restriction sites is useful in
cloning a foreign gene into the
plasmid.
5.
identify
the site of a point mutation.
6.
determine
the nucleotide sequence of any region of a DNA strand
A
2.
What do you know about Shotgun Sequencing ?
-
Shotgun
sequencing is a method used for sequencing a very large
piece of DNA like BAC DNA .
-
Longer sequences are subdivided into smaller fragments that can be
sequenced separately . then they are re-assembled to give the overall sequence
-
To
sequence a BAC : we take millions of copies of it and chop them all up randomly
then insert those into plasmidsthen grow lots of each one in bacteria and
sequence the insert .then we will able to reconstruct the sequence based on the
overlapping fragments we have sequenced .
3.
Summarize the essential features of a sanger DIDeoxy sequencing
experiment indicating the components of Sanger reaction ?
The essential features of a sanger DIDeoxy
sequencing experiment
a. Most popular method.
b.
Simpler and quicker allowing large output.
Within an hour the primer-annealing and sequencing reactions can be completed.
Stages:
1.
The DNA to be sequenced is called the
template DNA (one of the single
strands which was denatured using NaOH),
2. A
synthetic 5’-end-labeled oligodeoxynucleotide is used as the primer.
3. The
template DNA is hybridized to the primer.
4. The primer elongation is performed in
four separate polymerization reaction
mixtures. Each mixture contains
- 4 normal deoxynucleotides (dNTPs) in higher concentration
- a low concentration of the each of the 4 ddNTPs.
5. There is initiation of DNA synthesis by
adding enzyme DNA polymerase since the enzyme cannot distinguish between the
normal nucleotides and their analogues.
6.
The strand synthesis continues until a
ddNTP is added. The chain elongation ceases on the incorporation of a ddNTP because it lacks a
3’-OH group which prevents addition of the next
nucleotide.
7. There is a result of mixture of terminated fragments, all of different
lengths.
8. Denature DNA fragments.
9. Each of the four mixtures are run
together on a polyacrylamide gel for electrphoresis.
10. The separated fragments are then visualized by autography.
11. From the position of the bands of the resulting autoradiogram,
the sequence of the original DNA template strand can be read directly.
4. What are the uses of DNA fingerprints ?
A.
Diagnosis of
Inherited Disorders :
DNA fingerprinting is used to diagnose inherited disorders
in both prenatal and
newborn babies in hospitals around the world.
B. Developing Cures for Inherited Disorders
identify DNA patterns associated with the disease in question By
studying the DNA fingerprints
of relatives who have a history of some particular disorder,or by
comparing large groups of
people with and without the disorder
C. Biological Evidence
Such as DNA
fingerprints were used to link suspects to biological evidence -
blood or semen stains, hair, or items
of clothing – found at the scene of a crime
D. Personal Identification
Such as identify
casualties or persons missing in action by collect DNA fingerprints from all personnel .
5.
What are the bases for using blotting techniques ?
Visualization of specific DNA, RNA and protein
among many thousands of contaminating molecules requires the convergence of
number of techniques which are collectively termed Blot transfer .
Types of blotting techniques :
a.
Southern
blotting ( to detect DNA )
b.
Northern
blotting ( to detect RNA )
c.
Western
blotting ( to detect protein )
-
The
initial separation of molecules is done on the basis of molecular weight .
-
In general, the process
has the following steps, detailed
below:
a. Gel electrophoresis b.Transfer
to Solid Support c. Blocking
d. Preparing the Probe e. Hybridization f.
Washing
g. Detection of Probe-Target Hybrids
6.
Give short notes about :
also called as non-coding strand or templet
strand . this strand act as templet for
the synthesis of mRNA and which is complementary to it.
2. AUTORADIOGRAPHY - A
process to detect radioactively labeled molecules (which usually have been
separated in an SDSPAGE or agarose gel) based
on their ability to create an image on photographic or X-ray film.
3. BACK MUTATION -
Reverse the effect of a point or
frame-shift mutation that had
altered a gene; thus it restores the wild-type phenotype
4. CODON BIAS
The tendency for an organism or virus to use
certain codons more than others to
encode a particular amino acid
5. GENOMIC LIBRARY - A DNA
library which contain DNA fragments hopefully representing each region of the
genome of an organism
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