Isolating, Cloning and Sequencing DNA

Applications Topic: Isolating, Cloning and Sequencing DNA
Section B: Free Response Questions
1. Discuss
a) the aims of the human genome project. [6]
• to map all human genetic markers, i.e. by identification of their specific chromosomal location
• to construct a detailed physical map of the entire human genome
• to determine the base sequence of all 24 human chromosomes
• to develop technology for the management of human genome information
• to serve as an umbrella for similar mapping and sequencing projects on the genomes of other organisms, e.g. E.coli, yeast

b) the benefits of the human genome project. [8]
• improved diagnosis of disease and predisoposition to disease by genetic testing
• better identification of disease carriers through genetic testing
• better drugs can be designed using knowledge of protein structure
• greater possibility of correcting genetic disorders using gene therapy
• greater knowledge of family relationships through genetic testing, e.g. paternity testing in family courts
• advances forensic science through analysis of DNA at crime scenes
• improved knowledge of relationships between human and other organisms, which will help to develop better, more accurate classification systems.

c) the ethical concerns that have arisen about the human genome project. [6]
• unclear whether third parties should have rights to genetic test results – legislation is needed to ensure that there is no discrimination on the basis of genetic information
• reproductive issues regarding use of genetic information in reproductive decision making and reproductive rights. Prenatal genetic testing could lead to genetic manipulation or a decision to abort based on undesirable traits disclosed by the tests.
• Treatment vs enhancement of humans – no clear distinction between medical treatment and enhancement
• Fairness in access to advanced genome technologies – difficult to ascertain who should benefit or it will results in major worldwide inequities
• Commercialisation of products – difficult to determine who own genes and other pieces of DNA and to ascertain if patenting DNA sequences will limit the accessibility and development into useful products. [N08/P3/Q4]

2. a) Distinguish between a genomic DNA library and a cDNA library [6]

Feature Genomic DNA library cDNA library
DNA fragment cloning of all DNA fragments representing the entire genome, including coding and non-coding regions contains DNA fragments representing only the coding region of a genome.
Introns both the introns and the non - transcribed DNA are included in the clones intron sequences have been removed by RNA splicing during the formation of the mRNA, and a continuous coding sequence is therefore present in each clone.
Method DNA is cut using RE 
DNA fragments are incorporated into vector mRNA is extracted from a particular cells then reverse transcribe to form complementary DNA before incorporated into vector
Frequency All genes are represented equally in the library Genes that are transcribed abundantly will be represented more frequently in the library
No. of clones Larger number of clones to screen Smaller no. of clones to screen (as it contains only the coding regions)
Regulatory sequences Presence of regulatory sequences Absence of regulatory sequences


b) Describe the properties of plasmids that allow them to be used as cloning vectors. [6]
• Plasmids are circular, double-stranded DNA (dsDNA) molecules.
a) small molecules, size: a few kb to more than 100 kb
 facilitates the vector’s entry into host cells and the biochemical manipulation of DNA.
b) contain an origin of replication.
 The vector can replicate itself and the inserted gene of interest.
 During cell division, at least one copy of the plasmid DNA is segregated to each daughter cell, assuring continued propagation of the plasmid through successive generations of the host cell.
c) possess several unique restriction sites.
 The vector can be cut to insert the gene of interest.
d) contain genetic/ selectable markers.
 These are often resistance gene, which confer some well-defined phenotypes on the host organism which enables selection.
 i.e. allow us to identify the host cells that has taken up the recombinant DNA molecule.

c) Outline the large-scale production of a named important protein by genetic engineering.
[8]
• DNA sequences encoding the A and B chains of human insulin were chemically synthesised
• Each gene were placed under the control of the strong lac promoter and a part of the -galactosidase structural gene in the plasmid
• Both recombinanat plasmids containing the two artificial genes were transformed separately into E.coli.
• The 2 artificial genes were expressed independently as fusion proteins, consisting of the first few amino acid of -galactosidase  for initiation of translation in bacteria
• Lactose is added to induce transcription from the lac promoter
• Insulin fragment and -galactosidase were separated by a methionine residue
• Insulin polypeptides could be cleaved from the -galactosidase fragment by treatment with cyanogens bromide
• Purified A and B chains were attached to each other by disulphide bond formation
[N09/P3/Q4]