pathology final exam revision

what are the purines present in DNA?

adenine and guanine

what are the pyrimidines present in DNA?

thymine and cytosine

what is the molecular difference between RNA and ssDNA?

cytosine is replaced with uracil

what is PCR used for?

detect/amplify specific DNA or RNA sequences

what are the ingredients of the PCR reaction mix?

• template DNA

• primers

• Taq DNA polymerase

• nucleotides

• enzyme buffer

what is a DNA template?

DNA that contains the target sequence

what are the steps in a PCR reaction?

denaturation, annealing, extension

what does denaturation entail?

denaturation of the template dsDNA to ssDNA at 95°C

what does annealing entail?

attachment of primers to the target ssDNA

what are primers?

short DNA segments which anneal to complimentary sequences to the target DNA; starting point of reaction

what are universal primers?

complimentary to conserved DNA sequences

what are specific primers?

complimentary to sequences specific to particular organisms

what are random primers?

have many homologous sites in a genome

what does extension entail?

replication of target sequence using Taq DNA polymerase

why is Taq DNA polymerase used in PCR?

isolated from volcanic bacteria – thermally stable

what does Taq DNA polymerase do?

synthesises complimentary DNA strand in 5’ to 3’ direction starting from double-stranded region (annealed primers)

why are nucleotides included in the PCR mix?

building blocks to construct new DNA sequences

why are enzyme buffers included in the PCR mix?

creates suitable environment for Taq DNA polymerase enzyme to function

how many times is PCR repeated?

30–40

why is gel electrophoresis used to analyse PCR product?

DNA is slightly negatively charged

what dye is commonly used in gel electrophoresis?

ethidium bromide

how is reliability ensured in PCR?

inclusion of control DNA and specific primers; if the control is amplified, the PCR worked

what is specificity in terms of PCR?

only the target sequence is amplified

what is real-time PCR?

incorporates fluorescent substances (dyes or probes) to monitor PCR as it occurs

what is reverse transcriptase PCR?

uses reverse transcriptase to create copy of RNA as dsDNA to allow for PCR analysis

what are the advantages of PCR?

• quick

• sensitive

• low cost (routine)

• not necessary to purify organism for diagnostic PCR

what are the disadvantages of PCR?

• high equipment costs

• high irregular screening costs

• requires lab and molecular biology experts

how are nucleotides harvested using cell lysis?

nucleic acid release through structure destruction, removal of impurities

what are dNTPs?

deoxyribonucleotide triphosphate; building blocks for DNA synthesis

what is genetic diversity?

the total number of genetic characteristics in the genetic makeup of a species

what is genetic variability?

tendance of gene characteristics to vary

what is conserved DNA?

sequences of DNA shared among organisms

what is the function of ribosomes?

protein synthesis

what is the difference between prokaryote and eukaryote ribosomes

prokaryotes contain 3 rRNA molecules, eukaryotes contain 4

which rRNA molecule is located separately from the others in eukaryote ribosomes?

5S

what is ribosomal DNA?

areas of genomic DNA that include genes coding for rRNA molecules

why shouldn’t rRNA molecules mutate?

essential genes; mutations lead to death

why can internal transcribing regions mutate without much consequence?

not essential, doesn’t transcribe DNA

why are ITS regions amplified for sequencing?

very variable, measure relatedness between organisms

what are the kinds of allelic variations?

• microsatellites/simple sequence repeats

• segmental insertions/deletions

• single nucleotide polymorphisms

what are microsatellites?

simple sequence repeats with 1-13 base pair repeats

how are microsatellites analysed?

if flanking regions are known, primers can be designed for them and the number of repeats can be compared

what are single nucleotide polymorphisms?

variation in DNA sequence; occurs commonly within a population

what is a non-synonymous SNP?

SNP that alters the amino acid coded for

who created DNA sequencing?

Frederick Sanger in 1977

what is sanger sequencing?

determination of the precise sequence of nucleotides in a sample of DNA

what are dideoxynucleotides?

synthetic nucleotides lacking the 3’ OH (hydroxyl) group

why do dideoxynucleotides prevent chain elongation?

hydroxyl group binds to next nucleotide phosphate group; no OH, nowhere for the phosphate to bind

what does sanger sequencing entail?

• ssDNA template supplied with both deoxynucleotides and tagged dideoxynucleotides

• chain elongation proceeds normally until dideoxynucleotide is inserted

• fragments separated longest to shortest and fluorescence colour is recorded

what is pyrosequencing?

sequencing by synthesis using enzymes

what does pyrosequencing entail?

synthesis of compliment strand to immobile ssDNA and the recording of each added nucleotide

what is the basis of pyrosequencing?

pyrophosphate is generated during DNA extension, binds with adenosine 5’ phosphosulfate to form ATP, which is used in conjunction with Luciferin and luciferase to generate light

what does next generation sequencing entail?

• nucleotide is added, everything else washed away

• check colour of fluorescence

• fluorescent and terminating groups on nucleotide cleaved off

• repeat