pcr - in vitro cloning - and genetic screening and counselling

2 ways dna fragments can be clones

in vivo or in vitro

in vitro meaning

dna cloned outside of an organism - in a lab

how can fragments of dna be amplified in vitro

the polymerase chain reaction

machine used in pcr

thermocycler

what type of dna polymerase is used in pcr

taq polymerase

why is taq polymerase used in pcr

from hot springs - evolved to have an optimum much higher than our dna polymerase

taq polymerase optimum temperature

72 degrees

purpose of primers in pcr

short sequence of single stranded dna - complementary to the start and end of dna fragment sequence

explain the first step of pcr

temperature is increased to 95 degrees to break hydrogen bonds and split the dna into single strands - denaturing

what happens in pcr after the temperature is increased to 95 degrees and the dna is split into single strands

temperature is decreased to 55 degrees so that primers can attach - annealing - hydrogen bonds reform

what happens after the temperature is lowered to 55 and primers attach

synthesis - temperature is increased to 72 degrees - dna polymerase attaches complementary free nucleotides via phosphodiester bonds to make a new strand to align next to each template

3 diiferent temperatures in pcr

95 to 55 to 72

3 advantages of pcr

automated - more efficient - rapid - 100 billion copies of dna can be made in hours - doesnt require living cells - quicker and less complex techniques needed

what are dna probes

short singles stranded pieces of dna that are labelled radioactively or fluorescently so they can be identified

what are dna probes used for

to locate specific alleles of genes and to screen patients fr heritable conditions, drug responses or health risks

why does the patients dna and the dna probe need to be single stranded

patients dna is mixed with the dna probes - if the patient has the allele, the dna probe will hybridise and the label indicates the presence

explain how a patients sample is used in dna hybridisation

sample is heated to make it sinle stranded, causes hydrogen bonds to break (denaturing) - patients simple stranded dna sample is mixed with dna probe and cooled - any complementary sequences can align and form hydrogen bonds - anneal with dna probe

what must be known in order to locate a specific allele and then create the dna probe

the dna base sequence

how can the dna base sequence be determined when locating specific alleles of genes

using dna sequencing techniques ie sanger method

2 types of label used in dna probes

radioactive nucleotide or fluorescent lable

what happens to the dna after hybridisation

dna is washed - any unbound dna probes are washed away - label can be identified

one advantage to genetic screening

personalised medicine - some painkillers are more effective depending on genotype - also determine dose

benefits of personalised medicine

increased effectiveness, safety and saves money

example of personalised medicine

vitaman e given to diabetics reduces risk of cvd but for others with different genotype it can increase the risk

role of genetic counsellor

type of social work - people have their family history researched to consider the likelihood of them carrying alleles linked to diseases, before starting a family or for general health

what are patients informed of in genetic counselling

any potential risks to themselves or future offspring if they were to carry the allele

why might screening for alleles linked to breast cancer be beneficial if there is a family history of it

if you find the allele is present you can be screened for tumours more frequently, reduce environmental risk factors or opt for a mastectomy