Distinguish between the intersimple sequence repeat (ISSR) AND SIMPLES sequencing repeat

(SSR) Marker techniques

ISSR genomic DNA amplified with a microsatellite complementary prime while SSR based
primers are usually is mers to 20 mers.
Amplified products are resolved on agarose gel using ethidium bromine staining or
pdyacrylamide gels by silver staining
ISSR markers occur between microsate ilite DNA SSR marker are micro-satellite DNA
ISSR contain mecleotides repeats of 100-300 micleotides while SSR contain micleotides
repeats of 1-6 micleotides.
ISSR recognize multiple loci while SSR recognize helerozygnous loci.
ISSR amplify random sequence of the genome while SSR amplify tandem repeats.
ISSR is dormant while SSR is codominant.
2. Describe the polymerase chain reaction (5marks)
It is the in ratio amplification of DNA by repeated cycles of strand separation and
polymerization.
PCR is carried out in a single tube kept in a thermal cylinder programmable to set alternate
heating and cooling.
DNA to be amplified, reagents, two types of oligomecleotide primers, deoxy-rebimecleotides
and tag polymerase are added into the tube.
The DNA to be amplified acts as template strand for DNA polymerazation .
The reagents include Tris-HCL buffer, magnesium Chloride and Potassium chloride.
The deoxy-rebimecleotides include dATP, dGTP, dCTP, dTTP
The thermal cylinder is programmed in such a way as to provide cycles of 94c for 1 minute to
the reaction tube.
3. In electrophoretic separation the rate of migration of linear DNA is proportional to its
molecular weight.
Explain why this is not true for RNA
(2marks)
RNA is single stranded and this tends to form very extensive secondary and tertiary structures
through internal base paveiling. The internal base paining is different giving rise to different
molecules which the rate of migration will not be proportional at to molecular weight.
b) Hons would you treat RNA so that it’s electrophoretic mobility is proportional to its
molecular weight (3marks)
RNA is treated with Glyoxal to avoid the formation of secondary and tertiary structures.
When Glyoxal is issued Glyoxal adducts are formed with amino grps of bases.
Glyoxalacated RNA has migration rate proportional to its molecular weight because no
formation of secondary and tertiary structures.
4. Explain the mechanism of translation (5marks)
Translation is the process of converting nucleic acid information into amino acids.
During translation sequence of mecleotides present on the RNA is translated into amino acids
sequence of proteins.
Translation is carried out by ribosome where both ribosomes and tRNA dock on a matured
mRNA transcript and select multiple enzymes in an energy-intensive process that uses ATP as
well as GTP.
The main components of translation include mRNA, tRNA, Ribosomes, enzymes and proteins
Translation precedes in three different the stages and every stage is associated with different
proteins
The stages include:
I) Initiation
II) Elongation
III) Termination
5. What are transposons And how are they significant in recombinant DNA (5marks)
It is a DNA sequence that Moves from one place to another within agenome
During the movement of transposon , it Replicates into two; one copy is retained at the original
site and the other copy is transferred to a new site in the genome
Significance of transposons
I) Transfer foreign gene from one place to another
II) Restructure a genome
III) To construct DNA for gene coloring
6. Describe the southern bot technique
In the technique probes are used to detect nucleic acid molecules with sequences
complimentary either in total or in part electrophoresis of DNA
Probe anneals to fragments basing on complementary in totality or in part
Restricted DNA is separated by electrophoresis and the denatured to single strand by soaking in
alkaline (NaoH)
Single strand DNA is transferred to positively charged mtrocellulose membrane
DNA relatively negatively charged adheres to the membrane and creates an imprint or blot of
DNA profile
Membrane can then be probed with DNA probes.
Expose the hybridized membrane to infulm then expose it
7. The following enzymes are widely used in recombinant technology. Describe their mode of
action (5marks)
I) Ribosomes Ti and Ribomuclease A
Ribomudease Ti – attacks pyrimidine’s resulting in nude sides
II) Deoxyribomiclease – it cleaves DNA randomly either single or double stranded DNA
III) Alkaline phosphatase -removes the phosphatase at the 5`ppp and (5`04)the formation of the
hydroxy 5`end(5`04)
IV) T4 polymicleotide kinase-Labeling of DNA
V)T4 DNA ligase joining cohesive and blunt ended DNA fragments
8. Distinguish between affinity chromatography electrophoresis as used in DNA separation
(5marks)
Affinity chromatography is used with hydroxyapatite matrix.
It is used to separate double structured DNA from single stranded DNA since hydroxyapatite
matrix has high affinity for double stranded DNA.
This technique can help determine the proportionality of ds DNA to ss DNA Electrophoresis the
gel used is agarose
DNA is subjected to the electric field and migrates in the gel to the positive pole
As DNA migrates it can be separation based on the DNA fragment size
Size markers are used to characterize fragments according to size.
SECTION B
9. a) How is an expression vector used to produce desired proteins in living cells (5marks)
Expression vectors produce pure protein encoded by foreign genes.
They have a strong pkavyotic promoter, a foreign gene, a translation termination signal and
transcription stop signal
The rDNA transcribes into mRNA of the foreign gene alone
This results in the synthesis of pure proteins of the cloned gene.
b) Compare and contrast the efficiency of four widely used vector systems in DNA technology
(10marks)
Plasmids carry genes encoding resistance to antibiotics which be used as selectable markers.
Also plasmids can propagate independently in the cost
Plasmids can be present in multiple copies per cell thus increasing the amount of DNA that may
be isolated from a population
Plasmids have unique restriction sites. They have been modified to have more than 20
restriction sites within a small region. This allows a wide arrays of restriction enzymes to be
used to cut the target DNA
Bacteriophage have a system which they introduce foreign DNA into bacteria cells
Bacteriophages also accommodate larger DNA fragment than the plasmids up 25kb
Lambda bacteriophage has been restocked over the years and numerous derivatives that contain
only one or two sites for a variety of restriction enzymes
Bacteriophage lambda can be reconstituted in a test tube i.e vitro packaging
Cosmids can be used to construct genomi libraries
Since cosminds are packaged in phage strict ares, it allows the foreign DNA to be inserted into
the bacteria using transduction
Yeast artificial chromosome (YAC) can be used to get eukaryotic protein products with post
trans lational modifications.
Development of YACs has made it positive to put very large pieces of DNA into yeast cell and
reproduce them in large quantities
10 a) List five examples of gene regulation in eukaryotes and briefly discuss each (10marks)
In eukaryotes cells, the ability to express biologically active proteins comes under regulation at
several points
1. Chromatin structure; the physical structures of DNA, as it exists compacted into
chromatin, can affect the ability of transcriptional regulatory proteins (trans-cription
 factors) and RNA polymerases to find access to specific genes and actrirate transcription
from them.
2. Transcriptional imitation- factor that exert control include:-
a) Strength of promoter elements within the DNA sequences of a given gene,
b) Presence or absence of the enhancer sequences and the interaction between
multiple activator proteins and inhibitor proteins
3. Transcription processing and modification Eukaryotic mRNAs must be capped and
polyadenylated and introns must be accurately removed several genes have been
identified that undergo tissue specific patterns of alternative specific patterns of
alternative splicing, which generate bidogically different proteins from the same gene
4. RNA transport - A fully processed in RNA must leave the nucleus in order to be
translated into proteins
5. Transcript stability -unlike prokaryote in RNAs, whose half- lives are in the range of 1-5
minutes, eukaryotic mRNAs can vary greatly in their stability. Certain unstable
transcript have sequences that are signals for rapid degradation
6. Translational initiation-since many mRNAS have multiple methiomine codons, the
ability of ribosomes to recognize and initiate synthesis from the correct AUG codons
can affect the expression of a gene product
7. Post-translational modification include glycosylation, acetylation, fally acylation
disulfide bonds formations
8. Protein transport
In order for proteins to be biologically active, following translation and processing they
must be transported to their site of action
9. Control of protein stability.
Many proteins are rapidly degraded, whereas others are highly stable specific amino acid
sequences in some proteins have been shown to bring about rapid degradation
Gene regulation in prokaryotes
In bacteria, genes are clustered into operons: genes clusters that encode proteins necessary
to perform corditionally function
RNA that is transcribed from prokaryotic operons is polycistronic i.e multiple proteins are
encoded in a single transcript.
In bacteria, control the rate of transcriptional initiation is the predominant site for control of
gene expression
Initiation is controlled by two DNA sequence elements that are approx. 35bases and 10bases
respectively , upstream of the site of transcriptional initiation hence identified as -35 and -10
positions
These sequence elements are called promoter sequence because they promote recognition of
transcriptional start sites as RNA polymerase
The activity of RNA polymerase at a given promoter is regulated by interaction with
accessory proteins, which affect its ability to recognize stats sites
The regulatory proteins can act both positively (activator)and negatively (repressors)
Accessibility of promoter regions of prokaryote DNA is regulated by the interaction of
proteins with operators
Operator region is adjucent to the promoter elements
II) give an example of each for restriction enzymes that give “blunt ends”-
Alul,Haelll,Ball,Sau3 al “sticky ends”-EcoR1,Bam H1,Hind III
(B)5’GatCC3’
3’CCTAG5’
5’-CTGCA-3’
3’-ACGTC-5’
(C) In the presence DNA polymerase and the four DNATPS, the cat molecules will be
modified by adding complementary on the strands in growing direction however the extra
adenine base will be added to the three and of the growing strand
(D) Incubating the DNA agreement with T4 DNA ligase, the end of duplex DNA will be
joined whatever will not add any to a gap in the DNA
(E) Respective Bam H1 and Pst1 sites will be regenerated
It can not add any rudeotide to a gap created during restriction
It seals the Rick by establishing a covalent bond between 5 phosphate group and 3OH at the
nick.
The enzymes never seals the nick if there is no 5- phosphate group or if one more
nucleotides are missing.
BSLT
1. A) what is chromatography? (1mk)
It is a technique used used separate molecules in a mixture added to the solid or liquid
stationary state when travelling with the aid of a mobile phase.
B) explain how a thin layer chromatography works (4marks)
Is a technique used to separate non volatile mixtures.
 The experiment is conducted on a sheet of aluminium foil, plastic or glass which is
coated with a thin layer of adsorbent material caluminiuon oxide, cellulose or silcagel
On completion of the separation each component appears as spots separated vertically.
Each spot has a retention factor (Rf) expressed as:
Rf= distance travelled by a component divided by distance travelled by solvent
2. Explain plant tissue culture technique
It’s a collection of techniques used to maintain or grows plant cells tissues or o.gans under
sterile conditions on a nutrient culture medium of known composition
Plant tissue culture relies on the fact many plant cell are tolipotent
Single cells, plantcell without cell walls (protoplasts) pieces of leaves, system or roots can
often be used to generate a new plant on culture media given the acquired nutrients and
plant hormones
Different combinations of plant hormones are used to make the cells first divided rapidly
Cell need to be grown in sterile (aseptic) conditions to avoid fungus infection
3. Explain why animals are used in biomedical research (5marks)
Animals are biologically similar to human
Anatomical physiological and genetics similarities with human
Animal are susceptible to many health problems as humans
Animals have shorter life cycle thsn humans and as a result they can be studied throughout
their whole life span or a cross several generations
Animals can be easily maintained and bred in controlled environment (diet, temperature,
lighting)
4. Explain the principle and application of SDS -PAGE(5marks)
Sodium dodecy I sulfate polyacrylamide get electrophoresis is used for the
determination of molecular weight of individual polypeptide .
The protein mixture is first treated with a reducing agent dithionite to break the disulfide
bonds the SDS is added to disrupt nearly the non-covalent interaction in the protein,
unfolding the polypeptide chain.
SDS binding also imparts a large negative charge
On electrophoresis the large molecules move slowly compared with the small molecules
Migration distance are inversely proportional to the logarithmic of the molecular mess
5.Describe the difference buffer available for electrophoresis of native, double stranded
DNA(5marks)
Several different buffer are available for electrophoresis of native, double standard DNA
These contain tris-acetate and EDTA(PH 8:0;TAE)also called E buffer
Tris-borate (TBE)or
Tris-phosphate (TPE)at a concentration of 50mls(PH 7.5-7.8) Electrophoresis buffer are
uselly made up of concentration solution and stored at a room temperature
All these buffer work well, and the choice annoy them is largely a matter of personal
preference
TAE has the lowest buffering capacity of the three and will become exhausted if
electrophoresis is carried out for prolonged period of time.
6.Describe the polymerase chain reaction (5marks)

7.Describe a typical ELISA strategy for anti body screening (5marks).

Court purified autigen an immunoassay (pohysty Rene) in umbonate buffer.
Wash and block the plate well with appropriate regents
Add one to several dilution of immune serum to test wells and normal (pre-
immure)serum to other well, cover and inculcate the plate for 1 hour to allow antibodies
to bind
Wash the plate wells .Add an appropriate enzyrne conjugated detective secondary
antibody, and inculcate the plate for I hour to allow bindip to occur usually the goal is to
select for 1G, in which case the appropriate secondary antibody for mouse serum
samples would be an anti-mouse 1gG
Finally wash the plate and select active conjugated enzyme by adding the appropriate
substrate,such as TMB for an HRP-conjugated
8.Discuss the soul have blot teaching (5marks)
9.Discus the principle of microarray and their application in gene expression studies (10marks)
Microarrays are arrays of molecules immobilized at discribe locations on an inert surface
allowing them to be studied simultaneously microarray technology has become widely popular
since it is concept ually simple,easy to implement .
DNA microarray which have DNA molecules immobilized at precise location on glass or
silicone surface
Have become well accepted platforms for staying gene expression and mutation detection
Protein microarray, including ones in which antibodies are immobilized are expected to
revolutionized proteomic
Blotting technique here used hybridized between complementary nucleic acid molecular for
detection of DNA/RNA for quite a long time.
Microarrays can be considered on high throughput variants of the conversation
southern/northern hybridization
The molecules attached to the arrays is called the target the probes are arranged an spots on the
array gene expression
DNA microarrays have most widely used for studying gene expression
The mRNA isolated from cells under the conditions being studied for instance treatment with a
dung or toxic agent
Simultaneously mRNA from a reference untreated samples is prepared for comparison
cDNA prepared from both samples is labeled using different florescent dyes(say red or green)
The cDNA molecules are allowed to hyloriclize to the brobe molecules on the array
Each spot on the array corresponding to say a gene would therefore hybridized to cDNA
molecules tagged with red dye and green dye. The rotation of red:green fluorescence is a
measure of the relative levels of the gene in the test and reference samples.
Gene expression profile of cancer tissue have been used to classify the type of cancer and in
prediocting the prognosis of the cancer .
It has also been proposed that genes expression profiles can be used to identify toxic
compounds in complex mixtures by virtue of unique gene sets induced by them.
Gene expression profiling is definitely increased our knowledge of molecular events in a variety
of diseases.
b) Describe the DNA isolation steps (5 mark)
Lysing the cell – this is to help release the nudeic acids.
-Detergent dissolve the lipid membrane of cells.
-Enzymatic digestion to remove the cell wall of Gram+ bacteria and plant cells .
Physical disruption – grinding or homogenizing to remove cell wall.

2. Remove contaminating materials from nucleic acids

Enzymatic digestion – proteinases digest proteins.
Organic solvent extraction - proteins but not nucleic acids, dissolve in phenol and chloroform.
Chromatographic methods – DNA but not other components, binds to the material of choice.
3. Purification/concentration of nucleic acid .
Precipitation with alcohol – DNA is insoluble in either ethanol or isopropanol
Centrifugation – centrifugal force causes DNA to form pellets in bottom of the tube .
Dialysis – concentrates and cleans DNA by removing salts and other impurities that are small
enough to migrate through dialysis membrane.
4 .Differentiation of nucleic acid Genomic DNA and plasmoid DNA
Cesium chloride gradients – plasmids DNA and genomic DNA different in density.
Genomic DNA is less dense than plasmids DNA
Alkaline lysis- Alkaline conditions denature DNA
10.Discuss the principle procedure and applications of flow cytometry (5 marks)
Flow cytometry is a comprehensive technology used in cell biology to analyze single cells on
multiple parameters. The lasers in the device procedure scattered and fluorescent lights signals
detected by photodetectors.

Principles.
Flow cytometry runs on the principles of light scattering, excitation and emission. Florescently
tagged cell components gets excited when they pass through a laser beam, producing lights of
different wave lengths.
The fluorescence is used to analyse cellular properties.
Cells are analyzed by the following parameters
1. Visible light scatter; the forward scatter helps measure the size in the forward direction.
2. Fluorescence: cells can emit fluorescence from dyes , such as presidium iodide , reporter
genes focused to fluorescent proteins like GFP.

Procedures.
Sample preparation – Cells or tissues are harvested and prepared on a single cells
suspension.
Staining with a variability dye .
Using viability dyes allow to distinguish between live and dead cells and exclude the
dead ones during data acquisition and analysis .
Fixation and permeabilization .
When staining intracellular target, fixation is required to preserve the structure of
intracellular proteins.
Permeabilization disrupts the cell membrane.
Blocking- Prevents the non- specific binding of antibodies to cells.
Antibody incubation – cells are stained with fluorophosphate conjugated antibodies for
direct or indirect detection.
Detection and data analysis.
After antibody incubation , the experiment can be run in the flow cytometry.

Applications

Proteomics in early discovery

Assays can assess dozens of cell surface or intercellular proteins simultaneously.
Immunophenotyping for cellular composition.
Flow cytometry informs on the composition of a cell population based on lineage
market. This helps uncover cellular heterogeneity and rare cell populations that may be
valuable for future study
Gene expression analysis in cell signalling pathways
The technique can highlight gene expression tied to important cell signalling pathways.
Stimulated cells can be analyzed for up or down regulation of specific genes hence
regulatory mechanisms can be hypothesized
Disease diagnosis and monitoring in clinical labs.
Disease diagnosis and monitoring can be conducted via flow cytometry in clinical labs.
This is relevant for cancer and infectious disease where rapid testing is required
II) Discuss shotgun method of DNA sequencing and applications of DNA sequencing
(15marks)
This technique is useful for sequence genomes which are small , contact like those of
bacteria
It involves the shearing genomic DNA into fragments of 1kb average size and cloning
the fragments into plasmids vector.
DNA prepared from individual recombinant DNA colonies is separately sequenced on
sequenotors using the dideoxy method
In order to make certain that every single nucleotide in the genome is captured in the
final genome assembly, about 30,000 to 40,000 separate recombinant clones are
sequenced
The approximately 30,000 random sequencing reads from the random genomics DNA
fragments are loaded into a computer and different programs used to assemble
overlapping DNA sequences.
Applications of DNA sequencing
To analyze protein structure ie it’s DNA sequence
Through DNA sequencing we can understand the function of a specific sequence and
the sequence responsible for any disease
We can delect any mutation through comparative DNA sequencing study
Kinship study
DNA fingerprinting
By knowing the whole genome sequence.