Q1. (2 points) RFLP refers to the variation in the length of DNA fragments generated
by restriction enzymes and the laboratory technique which uses this characteristic to
compare DNA molecules.
RELP depends on genetic (DNA sequence) variation in a population. The DNA sequence
variation creates differences in restriction cut sites. Due to high variation in human
population, for instance, no two individuals except identical twins have the same DNA
sequence. Digestion of genomic DNA by restrictions enzymes generates DNA fragments
of different lengths in each individual. The fragments generated by restriction cutting can
be detected by Southern blot using probe specific for particular locus or allele.
Applications:
 Criminal investigation
 Disease diagnosis
 Strain/species identification
 To establish paternity
Part A indicates that child X can be a biological offspring of a putative father. On the
other hand the RFLP pattern from Part B shows that child X and the putative father do
not share any band. The evidence from these two experiments indicates that the child X is
less likely the biological offspring of the putative father, as RFLP mismatch is more
conclusive than matching bands. Two individual can have the same band by chance,
especially if the locus analyzed is not highly variable in the population. In this case the
actual biological father of child X may share the 1 kb band generated by ReE A with the
putative father. On the other hand, a mismatch by chance occurs with low probability. In
this particular case, mutation in child X that alters ReE B site has to occur to results in
mismatch between the child and the father. The best way to verify with accuracy is to use
other restriction enzymes and probe other loci or alleles.
Q2. (2 points) Maxam-Glibert (chemical) method: this approach of DNA sequencing
is based on reagents that cut DNA at particular base. Partial cutting of end labeled DNA
with a reagent that cleaves at particular base results in population of DNA fragment.
Analysis of DNA fragments generated by G, A/G, C and C/T specific cutting reagents on
high resolution polyacrylamide gel will give a ladder of bands which correspond to the
DNA sequence.
Sanger (enzymatic) method: this approach is based on termination of DNA synthesis with
2`3` dideoxynucleotide (ddNTPs). Like dNTPs, ddNTPs can be incorporated into DNA
by DNA polymerase. However, ddNTPs lack 3`OH group necessary to form the linkage
to the incoming dNTP. So ddNTPs terminates newly synthesized DNA fragments at
specific bases (either A, C, T, or G) and these fragments are then separated on high
resolution polyacrylamide gel. The sequencing process involves: denaturation of doublestranded DNA, annealing of primers to complementary site on template DNA and
synthesis of DNA by DNA polymerase in presence of dNTPs and about 1% of ddNTP. 5`
end labeled primer can be used for sequencing or the DNA can be labeled during
synthesis using radio labeled or fluorescent labeled nucleotides.
Template DNA: 5` gc gag caa ggc ggc agt ata ggt ctt gaa ggt ata cgc tag agt aac 3`
Q3. a. (2 points)
 self ligated plasmid pET without the insert
 recombinant plasmid with insert in two different orientations
 self ligated gene X, ligation between two or more gene X,
 ligation between two plasmids
b. (1 points)
 self ligated plasmid with out the insert,
 rarely two plasmid ligated to each other
c. (1 points)
 Isolate the plasmid and determine the size
 restriction enzyme digestion,
 PCR
 Southern blot
 Sequencing…
d. (1



points)
restriction enzyme digestion,
PCR
and sequencing
4. Denature DNA- facilitate transfer and probing by hybridaztion (2 points)