Applied Genetics (Questions & Answers)

Questions

 On

Applied Genetics

Mahmoud Ahmed Abulfetoh

Explain shorty :

1.     DNA sequencing, its methods & application

2.     Enzymatic chain termination method by F. Sanger et. Al and its advantages & disadvantages .

3.     Steps and application of PCR

4.     Real time Quantitive PCR

5.     The purpose of gel electrophoresis & why isnot it needed in some DNA sequencing methods ?

6.     Difference between Northern blotting & southern blotting

7.     The essential difference between the two broad classes of experimental methods used to amplify  a DNA Sequence of interest

8.      What is meant by ? :

WESTERN BLOT - VECTOR - TRANSLATION - TRANSFORMATION -  TERMINATOR - Taq POLYMERASE

Answers

1.           DNA sequencing & its application

ü DNA sequencing = determining the nucleotide sequence of DNA.

ü Methods of DNA sequencing :

•         Chemical cleavage method  by A. M. Maxam and W. Gilbert

•         Enzymatic chain termination method   by F. Sanger et. al.

•         Pyrosequencing :  measuring chain extension by pyrophosphate monitoring

Application of DNA seAuencing technologies

1.    the polymerase chain reaction(PCR), a method which rapidly produces

             numerous copies of a desired piece of DNA, 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

2.                Enzymatic chain termination method  by F. Sanger et. Al  and  its advantages & disadvantages.

•         This method uses single-stranded DNA.

•         Also known as dideoxy sequencing method because it involves the use of analogue of normal nucleotide 2’,3’-dideoxynucleoside triphosphates (ddNTPs). These are chain terminating nucleotides lacking 3’-OH ends.

This method  is based upon the incorporation of ddNTPs into a growing DNA strand to stop chain elongation.

Stages:

1.The DNA to be sequenced is called the template DNA. It is prepared as a single-stranded DNA after being spliced into M13 vector DNA. Infected E. coli host cells release phage particles which contains single-stranded recombinant DNA that includes the sample DNA. This DNA sample is then extracted from phage for sequencing purpose.

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 and

          - 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.

Advantages

•         Most popular method.

•         Simpler and quicker allowing large output. Within an hour the primer-annealing and sequencing reactions can be completed.

Disadvantages

•         Yielding of poor results owing to secondary structure in the DNA as sometimes DNA polymerases terminate chain elongation prematurely.

•         The sequence is obtained not from the original DNA molecule but from an enzymatic copy. So, there is a chance of incorporation of wrong bases.

Sanger method

Chain termination method

3.           Steps and application of PCR

Polymerase chain reaction, PCR, is an efficient and cost-effective way to copy or "amplify" small segments of DNA or RNA

The purpose of PCR is to make a huge number of copies of a gene

There are three major steps in a PCR, which are repeated for 30 or 40 cycles.

1. Denaturation: At 94 C (201.2 F), the double-stranded DNA melts and opens into two pieces of single-stranded DNA.

2. Annealing: At medium temperatures, around 54 C (129.2 F), the primers pair up (anneal) with the single-stranded "template" (The template is the sequence of DNA to be copied.) On the small length of double-stranded DNA (the joined primer and template), the polymerase attaches and starts copying the template.

3. Extension: At 72 C (161.6 F), the polymerase works best, and DNA building blocks complementary to the template are coupled to the primer, making a double stranded DNA molecule.

4.  Real time Quantitive PCR

1.       Same as PCR, but measures the abundance of DNA as it is amplified.

2.        Useful for quantitatively measuring the levels of mRNA in a sample.

3.       Uses reverse transcriptase to generate cDNA for the template.

4.       Can also be used to quantitatively estimate fraction of DNA from various organisms in a heterogenous sample (e.g, can be used to measure abundance of different microbes in soil sample).

5.       Can be used to type SNPs if primer binding is stringent.

6.       Fluorescent dye, SYBR Green, is incorporated into PCR reaction.

7.       SYBR Green fluoresces strongly when bound to DNA, but emits little fluorescence when not bound to DNA.

8.       SYBR Green fluorescence is proportional to the amount of DNA amplified, detected with a laser or other device.

9.       Experimental samples are compared to control sample with known concentration of cDNA.

SYBR Green binds to double-stranded DNA and fluoresces

5.  The purpose of gel electrophoresis & why it isnot needed in some DNA sequencing methods

In some DNA sequencing methods, such as classical Sanger (dideoxy) sequencing, the object is to make DNA Strands that incorporate label in four base-­‐specific reactions and allow DNA synthesis to produce a series of Fragments of different lengths. The different DNA synthesis products need to be separated according to size by Using gel electrophoresis so that they can be analyzed and interpreted .More modern method s often use some Type of sequencing-­‐by-­‐synthesis in which the incorporation of labeled nucleotides is continuously followed while the DNA synthesis is taking place. In that case, there is no need for gel electrophoresis.

6.  Difference between Northern blotting & southern blotting

§  Southern introduced this technique of southern blotting. Northern blotting was introduced by Alwine.

§  Separation of DNA occurs in southern blotting, separation of RNA occurs in northern blotting.

§  Denaturation is not needed in northern blotting but it is required in southern blotting.

§  Nitrocellulose filter membrane is used in southern blotting but not in northern.

§  DNA-DNA hybridization occurs in southern blotting. RNA-DNA hybridization occurs in northern blotting.

§  Amino benzoyloxymethyl filter paper is used in northern blotting but not in southern.

 

7.   the two broad classes of experimental methods used to amplify  aDNA Sequence of interest

•DNA cloning : amplifying DNA by replicating DNA sequences within cells, using ahost cell’s DNA polymerase.

•The polymerase chain reaction: amplifying DNA by replicating DNA sequences within attest tube , using apurifid DNA polymerase

8.  WESTERN BLOT - Proteins are separated by SDSPAGE, then electrophoretically transferred to a solid-phase matrix such as nitrocellulose, then probed with a labelled antibody (or a

series of antibodies)

VECTOR - A plasmid, cosmid, bacteriophage, or virus which carried foreign nucleic acid

into a host organism.

TRANSLATION - The process whereby mRNA directs the synthesis of a protein molecule; carried out by the ribosome in association with a host of translation initiation, elongation and

termination factors. Eukaryotic genes may be regulated at the level of translation, as well as the level of transcription.

TRANSFORMATION - Multiple meanings. With respect to cloning of DNA, refers to the

transformation of bacteria (usually to specific antibiotic resistance) due to the uptake of

foreign DNA. With respect to eukaryotic cells, usually means conversion to less-restrained

or unrestrained growth.

TERMINATOR - A sequence downstream from the 3' end of an open reading frame that serves to halt transcription by the RNA polymerase. In bacteria these are commonly sequences that

are palindromic and thus capable of forming hairpins. Sometimes termination requires the action of a protein, such as Rho factor in E.coli.

Taq POLYMERASE - A DNA polymerase which is very stable at high temperatures, isolated from the thermophilic bacterium Thermus aquaticus. Very useful in PCR reactions which must

cycle repetitively through high temperatures during the denaturation step.