Exercises
Homework
Lecture 4
1.
Describe a plasmid prep.
2.
What is meat by the term “sticky end”?
Sticky end and blunt end are biological terms describing the two possible configurations resulting the
breaking of double-strain of DNA. DNA exhibits a stabilizing interaction between complementary base
pair, providing specificity to the pairing of two strands of DNA. If two complementary strands of DNA
are of equal length, then they will terminate in a blunt end.
3.
What is a genomic clone?
The process of producing, exact copies of a single gene, other DNA segments, an entire cell or a complete
organism. Cloned collections of DNA are called clone libraries and are useful tools in helping scientists
piece together our genetic information.
4.
What is cDNA clone?
Complementary DNA - single-stranded DNA that is complementary to messenger RNA or DNA that has
been synthesized from messenger RNA by reverse transcriptase.
5. What are the steps involved in marking a genomic library?
collection of vectors carrying different inserts that represent all or some of the genes from an
organism or cell. libraries are used to find individual genes within complex genomes
Steps:
1) Isolation of chromosomal DNA
2) Fragmentation of DNA
a)
Physical breakage - pipetting, physical agitation (vortexing), sonication
b) Restriction enzyme digestion
3) Isolation and ligation of DNA fragments
6.
Explain in a few sentences the importance and principle of PCR
A method for amplifying a DNA base sequence using a heat- stable polymerase and two 20-base primers,
one complementary to the (+)-strand at one end of the sequence to be amplified and the other
complementary to the (- )-strand at the other end. Because the newly synthesized DNA strands can
subsequently serve as additional templates for the same primer sequences, successive rounds of primer
annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the
desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.
A method for amplifying a DNA base sequence using a heat- stable polymerase and two 20-base primers,
one complementary to the (+)-strand at one end of the sequence to be amplified and the other
complementary to the (- )-strand at the other end. Because the newly synthesized DNA strands can
subsequently serve as additional templates for the same primer sequences, successive rounds of primer
annealing, strand elongation, and dissociation produce rapid and highly specific amplification of the
desired sequence. PCR also can be used to detect the existence of the defined sequence in a DNA sample.
7.
Explain in a few sentences the importance and principle of gel electrophoresis
The Electrophoretic methods are separating the molecules depending of their size. Electrical charges are
carrying molecules through an electrical field whereby the molecules (DNA fragments) are negatively
charged. Due to the negative charge of the DNA provided by phosphate groups the molecules are moving
towards the positive electrode. The electrophoresis is used to separate and analyse molecules on a
polymer matrix ranges in pore size from 1 to 1000 nanometers. The gel provides a certain resistance to
the molecules moving in the electric field. The following criteria influence the distance of separation:
• the distance between the particles
• the electric field strength
• the size of the molecules
• the conformation of the molecules
Usually two forms of gel are used:
• Agarose gel is purified Agar which is a natural long-chain polysaccharide produced from
seaweed. The agarose gel is produced by heating agarose powder in a buffer for gelelectrophoresis,
pouring it in a gel chamber and cooling it down.
• Polyacrylamide gel consists of the small synthetic acrylamide molecules and polymerizes
to long chains by the action of a catalyst. The pores are smaller than in the agarose gel
and is therefore used for the separation of proteins or also smaller nucleic acids.
8.
Expalin in a few sentences the importance and principle of southern blotting
The Southern blotting technique involves transfer of DNA fragments separated in electrophoretic gels to
membrane filters for detection of specific base sequences by complementary probes. Southern blots are
used to identify and quantitate specific DNA sequences.
9.
Explain in a few sentences the importance and principle of DNA sequencing.
The purpose of sequencing is to determine the order of the nucleotides of a gene. For sequencing, we
don't start from gDNA (like in PCR) but mostly from PCR fragments or cloned genes
1. The sequencing reaction :
There are three major steps in a sequencing reaction (like in PCR), which are repeated for 30 or
40 cycles.
1. Denaturation at 94°C :
During the denaturation, the double strand melts open to single stranded DNA, all
enzymatic reactions stop (for example : the extension from a previous cycle).
2. Annealing at 50°C :
In sequencing reactions, only one primer is used, so there is only one strand copied (in
PCR : two primers are used, so two strands are copied). The primer is jiggling around,
3.
caused by the Brownian motion. Ionic bonds are constantly formed and broken between
the single stranded primer and the single stranded template. The more stable bonds last
a little bit longer (primers that fit exactly) and on that little piece of double stranded
DNA (template and primer), the polymerase can attach and starts copying the template.
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.
extension at 60°C :
This is the ideal working temperature for the polymerase (normally it is 72 °C, but
because it has to incorporate ddNTP's which are chemically modified with a fluorescent
label, the temperature is lowered so it has time to incorporate the 'strange' molecules.
The primers, where there are a few bases built in, already have a stronger ionic
attraction to the template than the forces breaking these attractions. Primers that are on
positions with no exact match, come loose again and don't give an extension of the
fragment.
The bases (complementary to the template) are coupled to the primer on the 3'side
(adding dNTP's or ddNTP's from 5' to 3', reading from the template from 3' to 5' side,
bases are added complementary to the template)
When a ddNTP is incorporated, the extension reaction stops because a ddNTP contains
a H-atom on the 3rd carbon atom (dNTP's contain a OH-atom on that position). Since
the ddNTP's are fluorescently labeled, it is possible to detect the color of the last base of
this fragment on an automated sequencer.
2. Separation of the molecules :
After the sequencing reactions, the mixture of strands, all of different length and all ending on a
fluorescently labelled ddNTP have to be separated; This is done on an acrylamide gel, which is capable of
separating a molecule of 30 bases from one of 31 bases, but also a molecule of 750 bases from one of 751
bases. All this is done with gel electrophoresis. DNA has a negative charge and migrates to the positive
side. Smaller fragments migrate faster, so the DNA molecules are separated on their size.
3. Detection on an automated sequencer :
The fluorescently labelled fragments that migrate trough the gel, are passing a laser beam at the bottom of
the gel. The laser exites the fluorescent molecule, which sends out light of a distinct color. That light is
collected and focused by lenses into a spectrograph. Based on the wavelength, the spectrograph separates
the light across a CCD camera (charge coupled device). Each base has its own color, so the sequencer can
detect the order of the bases in the sequenced gene.
4. Assembling of the sequenced parts of a gene :
For publication purposes, each sequence of a gene has to be confirmed in both directions. To accomplish
this, the gene has to be sequenced with forward and reverse primers. Since it is only possible to sequence
a part of 750 till 800 bases in one run, a gene of, for example 1800 bases, has to be sequenced with
internal primers. When all these fragments are sequenced, a computer program tries to fit the different
parts together and assembles the total gene sequence.
http://homepages.strath.ac.uk/~dfs99109/BB211/GenomicLibrary.html
http://www.thefreedictionary.com/complementary+DNA
http://www.hyperdictionary.com/dictionary/polymerase+chain+reaction
http://allserv.rug.ac.be/~avierstr/principles/seq.html
http://genome.tugraz.at/Theses/Thumser2003.pdf