Molecular Techniques in Biology

Question 1

describe PCR

Answer 1

• heating to 95 degrees causes the H bonds between base pairs to break, forming single-stranded DNA
• cooling to 55 degrees allows the forward and reverse primers to anneal to the complementary sequences on the 3’ end of the single-stranded DNA through the formation of hydrogen bonds
• heating to 72 degrees allows for primer extension and is the optimum temperature for Taq polymerase
• Taq polymerase binds to the 3’ end of primers and adds deoxyribonucleotides that are complementary to the DNA template and catalyses the formation of phosphodiester bonds beween adjacent deoxyribonucleotides

Question 2

explain the use of PCR

Answer 2

• amplifies specific sequence of DNA within a relatively short time
• with sufficient amount of DNA, tests for detection of bacteria can be carried out
• the amount of DNA amplified within a specific duration can allow for determination of viral concentration
• can be used in the analysis of human DNA e.g. in DNA fingerpringing for paternity testing/criminal investigation
• can clone genes which can then be used in genetic manipulation
• can detect genetic defects in early stages/mutations that cause a disease or condition
• used to amplify and study ancient DNA fragments from fossils
• can be used to classify organisms by comparing similarities in DNA sequences of different organisms to verify evolutionary relationship

Question 3

explain the advantages of PCR

Answer 3

• produces large numbers of copies of DNA for analysis in a relatively short span of time
• cell-free method of DNA replication hence requires no cleanup of unwanted cellular debris/vector DNA
• able to efficiently amplify targets up to 35kb in length with high fidelity and high yield

Question 4

explain the limitations of PCR

Answer 4

• need to first know the nucleotide sequence of one short DNA sequence flanking in order to synthesise the PCR primers
• highly sensitive, could amplify sequences that are not the target sequence if the sample is contaminated with other DNA molecules with sequences complementary to the primers used
• amplified DNA copies might have changes in their nucleotide sequences due to errors that occur at low but significant frequencies
• Taq polymerase does not contain a built-in proofreading activity hence it produces a higher than normal frequency of replication errors

Question 5

briefly describe the principles of gel electrophoresis

Answer 5

• electrical current applied across gel causes negatively charged DNA to move to positively-charged electrode
• agarose gel acts as molecular sieve for DNA fragment
• smaller fragments move faster and hence will travel further in a fixed period of time than larger fragments
• resulting in separation of fragments by size/length/number of electrodes

Question 6

describe what happens during nucleic acid hybridisation

Answer 6

• at high temperatures, hydrogen bonds are broken between complementary bases, separating double-stranded DNA
• lowering temperature allows annealing of DNA probes to single-stranded DNA to form hybrid double-stranded DNA

Question 7

describe gel electrophoresis

Answer 7

• DNA fragments are placed in wells at the negative electrode end of an agarose gel
• DNA is negatively charged and moves towards the positive electrode when an electric field is applied
• agarose gel acts as a molecular sieve for DNA fragments
• distance travelled in a given time depends on the molecular weight of the DNA/larger DNA fragments travel slower and smaller fragments travel faster
• as DNA is invisible to the naked eye, a small amount of loading dye is added to give an indication of the distance that DNA bands had travelled
• DNA ladder is used to estimate the size of DNA fragment in the separate bands
• ethidium bromide can be added to visualise all the fragments under UV light

Question 8

describe southern blotting

Answer 8

• DNA fragments are transferred from gel to nitrocellulose membrane by capillary action
• radioactively-labelled DNA probes are added to the membrane which binds to complementary DNA fragments
• by autoradiography, the positions of bound probes are visualised using photographic film
• giving rise to dark bands on a light background

Question 9

explain the use of gel electrophoresis and southern blotting

Answer 9

• allows identification of different alleles/mutations of genes
• allows DNA fingerprinting
• allows sequencing of DNA
• allows purification/isolation of target DNA