PCR

Question 1

Steps of PCR and temperature that each occurs at

Answer 1

STEP 1: DENATURATION 94º C
Target DNA is denatured and the DNA strands are separated and
are ready for the next step

STEP 2: ANNEALING 55º C
2 short oligonucleotide primers are annealed to the denatured DNA. The primers are complementary to the two 3’ ends of the DNA to
be amplified.

STEP 3: EXTENSION 72º C
The primers are then extended by DNA polymerase in the
presence of the four dNTPs, two duplex DNA copies are
generated from the template

Question 2

Applications of PCR

Answer 2

▪ Production of probes. Radioactive PCR to create a labeled fragment.
Can be used in hybridization experiments
▪ Gene cloning and manipulation. Amplify a gene + restriction enzymes sites
in the 5’ ends from genomic DNA and ligate to a vector
▪ Gene mutagenesis. Incorporate mutations by using primers with the desired
mutation.
▪ DNA sequencing. By amplifying just one strand, using one primer. Modified
method of Sanger
▪ Amplification of ancient DNA. Modern techniques to extract tiny amounts of
DNA from dried tissues or tissues embedded in amber allow to study museum
specimens and archeological samples
▪ Diagnostic in medicine. Bacteria and viruses can be readily detected.
▪ Forensic DNA typing. Determine paternity and whether DNA biological
samples are from a suspect. Tiny amount of sample needed from a hair, few
sperm, skin or blood. An individual DNA profile is highly distinctive because
many genetic loci are highly variable within a population. Amplifying different
locus you can distinguish one person from another

Question 3

What are alu elements? [length, location, how are they replicated?]

Answer 3

Alu elements

-300 bp sequences containing Alu I cleavage
sites.
-found in exons but mostly exist in introns and
other non-coding regions.
-Replication is through an RNA intermediate,
copied into ds DNA called retrotransposon that inserts randomly elsewhere in the genome

Question 4

homozygous insertion vs heterozygous insertion vs no alu homozygous insertion

Answer 4

-for this experiment, pcr is used to amplify the locus. Primers are annealed to the sides of the gene of interest

homozygous insertion (both strands have the 300 bp insertion)

heterozygous insertion (only one strand has the 300 bp insertion)

no alu homozygous insertion (no insertion on both strands

Question 5

Reagents for PCR experiment

Answer 5

-10X PCR Buffer (10uL)
-yeast DNA template (10ng)
-primer (20 pmol each)
-BSA (10ug)
-5uL of 1 mM dNTP working stock solution (50uM each dNTP)
-2 units Taq polymerase
-distilled water (to 100 uL)