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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 :

1.   ANTISENSE STRAND ( or primer )
   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.

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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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