Medical Interventions 2.1.2

How does a thermal cycler help the process of PCR?

- It uses a Peltier block to heat and chill the tubes very rapidly. - It does it over and over again through the PCR, that's why it's called thermal cycler (thermal = heats and chills, cycler = over and over).

How can a scientist target a gene of DNA in a PCR reaction?

- Use a positive control and a negative control, to make sure that the primers work. - The PCR is done by mixing the primers, dNTPs, buffer, and Taq Polymerase in premix tube, aliquotting small amounts into test tubes, and then adding the test DNAs (positive control or negative control, or a unknown sample). - The test tubes are then placed into a PCR machine that will heat up and cool down according to a standard program, for a number of cycles. - After the PCR, the scientist can test if the reaction worked by running the reaction on an agarose gel.

Explain how the process of PCR can be used in the identification of a disease pathogen?

- in PCR they'll amplify the DNA sequence of the pathogen , then after sequencing , they'll identify the identity of this DNA and identify the pathogen by comparing its genes with the gene bank .

Brainstorm how you could run a PCR reaction if you did not have access to a thermal cycler?

- three heat sources with annealing, elongation and denaturation temperatures, a clock and then put your cups manually from one source to the next for the needed time...water bathes or heat blocks are possible as heat sources....

Steps of PCR

1. Denaturation 2. Annealing 3. Extension

Taq Polymerase

A DNA synthesis enzyme that can withstand the high temperatures of PCR

How do you think PCR can be used to diagnose genetic diseases and disorders?

A PCR test (combined with gel elect.) can confirm the presence of a suspected pathogen by using long primers specific to the pathogen of interest. You're just testing for the presence or absence of an amplicon.

Restriction Enzyme

A degradative enzyme that recognizes specific nucleotide sequences and cuts up DNA

Primer

A short piece of DNA or RNA that is complementary to a section of template strand and acts as an attachment and starting point for the synthesis strand during DNA replication

polymerase chain reaction (PCR)

A technique for amplifying DNA (make copies of a specific DNA segment) in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides.

What are DNA Nucleotides or dNTDS? What do they do?

A,T,G,C and they are the building blocks for new DNA strands

Vector

An agent (as a plasmid or virus) that contains or carries modified genetic material and can be used to introduce extra genes into the genome of an organism

DNA translation

Process by which mRNA is converted into a protein

sticky ends

Single stranded ends of DNA left after cutting with enzymes

Why was it necessary to run a control in this experiment? What does the control tell you?

So you can compare and contrast the differences and similarities in an experiment.

Why do we use Taq polymerase and not regular human polymerase?

Taq polymerase is used as the bacteria can withstand high temp. which then allow it to withstand the melting step of PCR. Human polymerase would not be able to do this as it could not withstand such high temps.

Genome

The complement of an organism's genes; an organisms genetic material

genetic test

The use of methods to determine if someone has a genetic disorder, will develop one, or is a carrier

blunt ends

blunt ends Restriction fragments with no overlapping ends and that never combine with another type of DNA

What is supernatant?

clear liquid found on top of solid solution after a mixture has been centrifuged contains the inclusion bodies

Denaturation

hydrogen bonds are broken strands are separated; occurs at 95º C

SNP (single nucleotide polymorphisms)

mutations in genes that can lead to variation in traits or disease

Based on what you know about the process of PCR, what reagents must have been included in the PCR bead you used in the experiment?

nucleotides, taq polymerase, and primer

Annealing

primers bind to complementary DNA as temperature lowers 60º C

RFLPs

restriction fragment length polymorphisms

Extension

temp is raised to 72º C, new strand of DNA is made by adding taq polymerase