Techniques in Molecular Biology Flashcards
Why use molecular biology techniques?
Understand the genetic basis of disease requires the analysis of DNA
how do we target a gene or an exon?
Analyse or manipulate a single human exon or gene
Specifically recognise
a short DNA sequence of interest
Nucleotide hydridisation
Selectively amplify =a short DNA sequence of interest
Use DNA polymerases to make multiple copies of sequence
how many hydrogen bonds between a and T
2
how many between c and g
3
how can we break the h+ bonds in DNA
heating
can seperated DNA strands be re assocated again?
yes
Hybridisation?
association of any two complementary nucleic acid strands to form a double-stranded nucleic acid
what is the purpose of PCR
for amplifying areas of DNA
what are the componnets used for PCR
dna
primers
nuceleotides
buffers
dna taq polymerase
what is the point of primers in PCR
- single-stranded DNA that help DNA polymerase to initiate the synthesis of new DNA strands.
Primers are complementary to the target DNA sequence
WHAT IS THE POINT OF nucleotides in pcr
They act as raw material for new DNA synthesis
what is the point of a buffer in pcr
A salt solution used to stabilize the reaction components, especially the DNA, and maintain an optimal pH during the reaction
what is the pupose the enzy,e dna taq poymerase in pcr
The enzyme that synthesizes fresh DNA strands complementary to the target DNA sequence
why do we purposely use taq?
enzymeable to withstand the protein-denaturing conditions, required during PCR.
what does pcr increase
PCR increases the yield of DNA exponentially
what does pcr allow?
allows the in-vitro amplification of specific target DNA sequences using a single copy
Detection and identification of the PCR product is usually carried out by:
agarose gel electrophoresis
DNA sequencing
summarise Gel electrophoresis:
Separate DNA according to size
Molecules are pushed through a gel that contains small pores by an electric field
One end of gel has a +ve charge and other –ve.
DNA is –vely charged due to the phospate group, will be pulled toward the +ve end of gel.
Smaller molecules migrate more quickly and travel further
Visualisation of PCR products
Loading buffer is added to the PCR product and then loaded into wells
A marker is added to one well, ‘ladder’ with known size bands
Visualise DNA – gel is stained with a fluorescent dye that binds to DNA and can see when placed on a UV transilluminator
Size of DNA – imagine horizontal line running from ladder to DNA
what can go wrong when doing pcr
Does not amplify
Contamination
Non-specific priming. Primers are not specific to sequence intended and see wrong size product or banding effect
how to improve the pcr reaction
Redesign Primers
Increase annealing temperature
Alter number of cycles performed
advantages of pcr
Easy to set up (real-time PCR)
Fast turnaround time
disadvantages of pcr
Extremely liable to contamination
High degree of operator skill required
how is pcr used in pregnancy
For prenatal diagnosis, PCR used to amplify DNA from fetal cells obtained from amniotic fluid
what can pcr detect
-direct sequencing of DNA
-restriction enzyme analysis (RFLP)
Other advantages of PCR in forensic science are:
relatively simple to perform and simple to standardize
results obtainable within 24 hours.
