Molecular Techniques in Biology Flashcards
1
Q
describe PCR
A
- 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
2
Q
explain the use of PCR
A
- 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
3
Q
explain the advantages of PCR
A
- 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
4
Q
explain the limitations of PCR
A
- 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
5
Q
briefly describe the principles of gel electrophoresis
A
- 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
6
Q
describe what happens during nucleic acid hybridisation
A
- 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
7
Q
describe gel electrophoresis
A
- 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
8
Q
describe southern blotting
A
- 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
9
Q
explain the use of gel electrophoresis and southern blotting
A
- allows identification of different alleles/mutations of genes
- allows DNA fingerprinting
- allows sequencing of DNA
- allows purification/isolation of target DNA