Gene analysis

• Which end does DNA migrate to in gel electropheresis

  The anode
• Why does DNA migrate to the anode

  The sugar-phosphate backbone is negatively charged.
• What is the role of agarose gel in separation

  It slows the movement of large molecules
• How does electropheresis seperate

  By size and charge
• What is used to visualize DNA bands in electrophoresis

  Ethidium bromide and UV irradiation
• What is the resolution of electrophoresis

  It is the size range of molecules that can be separated
• What is the relashionship between the percentage of agarose and resolution

  As the percentage increases the resolution decreases
• What are the techniques used to study gene structure

  Restriction mapping

  Fragment length polymorphism

  Polymerase chain reaction

  Souther blotting

  DNA sequencing
• What does restriction mapping mean

  It is the process of mapping the positions of different restriction sites with a DNA molecule
• How is a restriction map generated

  DNA is cut with different restriction endonucleases.

  Gel electrophoresis determines the size and number of fragments produced.

  Double digests are then performed.

  The results are then compared and the sites are mapped.
• What is a double digest

  When DNA is cut with two restriction endonucleases
• What is the restriction map for these fragments

  24.5 and 15 are on the ends.

  9 is in the middle

  
• What is a restriction fragment length polymorphism

  This is a change in a DNA sequence that changes the restriction site
• What is used in the synthesis stage of PCR

  Taq polymerase and deoxyribonucleotide triphosphates.
• What are the tempreatures used in PCR

  94 for denaturing.

  55 for annealing.

  72 for synthesis.
• What are the requirements for DNA polymerase

  Deoxyribonucleotide triphosphates. It requires a free hydroxyl group on the 3' which is provided by primers.
• What is used in the annealing stage of PCR

  Primers
• Why is taq polymerase used in PCr

  It is heat resistant and so the reaction can be repeated without additional enzyme.
• What is southern blotting

  It is used to identify specific fragments within a mixture.
• Describe the process of southern blotting

  The DNA is digested with a restriction endonuclease and separated using gel electrophoresis.

  The DNA is then denatured and filtered with a salt solution.

  A radioactive or fluorescent probe is incubated with the DNA and detected using an X-ray.
• What is DNA sequencing

  The determinitation of the order of bases in a strand of DNA. It is written in the 5 to 3 direction.
• What type of DNA sequencing is the Sanger method

  Chain
• What is an example of chain sequencing

  The sanger method
• Briefly describe chain sequencing

  DNA synthesis is terminated early by the addition of chain-terminating dideoxynucleotides.
• Is a high or low concentration of dideoxynucleotdes used in chain sequencing

  low
• What is ddNTP

  Dideoxynucleotide triphosphate
• What are the requirements for chain seuqnecing

  Single-stranded DNA

  Primers and dNTPs.

  ddNTPs.

  DNA polymerase
• Describe sanger sequencing [chain] [Use abreviations such as dNTP]

  The 4 ddNTPs are labelled with different fluorescent dyes. When ddNTPs are incorporated in the DNA sequence, DNA synthesis stops. The DNA fragments are separated using electrophoresis and fluorescent light is emitted.
• Why do ddNTPs stop DNA synthesis

  They do not provide a free hydroxyl group on the 3 end for DNA polymerase.
• What sequence depth or coverage is used in chain sequencing

  A fivefold
• What does a five fold coverage mean in sequencing

  Each nucleotide is present in 5 separate reads
• What si the difference between cycle and chain sequencing

  Cycle sequencing uses taq polymerase which allows it to repeat with a smaller number of template DNA
• What type of blotting is used for DNA

  Southern
• What type of blotting is used for RNA

  Northern
• What type of blotting is used for proteins

  Western