8.4.2 Difference in DNA used in identification and diagnosis

• DNA probes

  ● Short, single stranded pieces DNA

  ● Base sequence complementary to bases on part of a target allele

  ● Usually labelled with a fluorescent or radioactive tag so can be identified
• Why are probes longer than just a few bases?

  ● A sequence of a few bases would occur at many places throughout the genome

  ● Longer sequences only likely to occur in target allele
• DNA hybridisation

  ● Binding of single stranded DNA probe to complementary single strand of DNA

  ● Forming hydrogen bonds / base pairs
• Locating specific alleles of a gene (genetic screening)

  1. Extract DNA and amplify by PCR

  2. Restriction enzymes cut DNA at specific base sequences (either side of target gene)

  3. Separate fragments by gel electrophoresis according to length
• Part two of genetic screening:

  4. Transfer to a nylon membrane and treat to form single strands with exposed bases

  5. Labelled DNA probe will hybridise with its target allele (then wash to remove unbound probe)

  6. To show bound probe either:

  ● Expose membrane to UV light if fluorescently labelled probe used

  ● Use autoradiography (expose to X ray film) if radioactive probe used
• Use of labelled DNA probes to screen patients for heritable conditions, drug

  responses or health risks

  ● Screening patients for heritable conditions

  ○ eg. cystic fibrosis → find out if a carrier

  ● Screening patients for drug responses

  ○ eg. alleles mean patients respond well to certain drugs and don’t

  respond to others as code for enzymes affecting metabolism of drugs

  ● Screening patients for health risks

  ○ eg. alleles that predispose to high blood cholesterol

  ○ eg. alleles that predispose to breast cancer
• Students should be able to evaluate information relating to screening individuals for genetically determined conditions and drug responses.

  • Positives:
  • Make informed reproductive choices
  • Make informed lifestyle choices
  • Identify heritable conditions early and use personalised medicine for treatment
  • Negatives:
  • Causes depression
  • Increase cost of health or life insurance
  • Can cause abortion
• What is gel electrophoresis?

  ● A method used to separate nucleic acid (DNA / RNA) fragments OR proteins

  ● According to length / mass (number of bases / amino acids) AND charge (DNA is negatively charged due

  to phosphate groups and protein charge varies based on amino acid R groups)
• Describe examples of the use of labelled DNA probes

  ● Screening patients for heritable conditions (eg. cystic fibrosis)

  ● Screening patients for drug responses (some alleles code for enzymes involved in drug

  metabolism that enable better responses to certain drugs)

  ● Screening patients for health risks (some alleles predispose patients eg. to high blood cholesterol)
• Describe the role of a genetic counsellor

  1. Explain results of genetic screening, including consequences of a disease

  2. Discuss treatments available for genetic condition

  3. Discuss lifestyle choices / precautions that might reduce risk of a genetic

  condition developing eg. regular screening for tumours or a mastectomy

  4. Explain probability of condition / alleles being passed onto offspring →

  enable patients to make informed decisions about having children
• What is personalised medicine?

  ● Medicine tailored to an individual's

  genotype / DNA

  ● Increasing effectiveness of treatment

  eg. by identifying the particular

  mutation / allele causing cancer and

  treating it with tailored drugs
• Evaluate the screening of individuals for genetically determined conditions

  and drug responses

  For ✓ Can enable people to make lifestyle choices to reduce chances of diseases developing

  ✓ Allows people to make informed decisions about having their own biological children

  ✓ Allows use of personalised medicines, increasing effectiveness of treatment
• Evaluate the screening of individuals for genetically determined conditions and drug responses

  • X Screening for incurable diseases or diseases that develop later in life (where nothing positive can be done in response) may lead to depression
  • X Could lead to discrimination by insurance companies / employers
  • X May cause undue stress if patient does not develop the disease
• Mistake

  *Stating that electrophoresis and DNA probes are used

  in genetic screening but not describing their roles.*

  Explanation

  Electrophoresis separates DNA fragments by length.

  DNA probes bind / hybridise with target alleles.
• Mistake

  *Confusing DNA probes with marker genes or primers.*

  Explanation

  Primers are used in PCR. Marker genes are used

  when creating genetically modified organisms.
• Mistake

  “The DNA sample is broken into smaller fragments through heating.”

  Explanation

  Restriction enzymes break the DNA into smaller fragments.
• Mistake

  “Radioactivity shows up when X-rayed.”

  Explanation

  Exposing radioactive probes to X-ray film

  (autoradiography) is not the same as X-raying.
• Explain how genetic screening can be used to locate specific alleles of genes

  1. Extract DNA and amplify by PCR

  2. Cut DNA at specific base sequences (either side of target gene) using restriction enzymes

  3. Separate DNA fragments / alleles (according to length) using GEL ELECTROPHORESIS

  4. Transfer to a nylon membrane and treat to form single strands with exposed bases
• Part 2 - Genetic screening, after Gel electrophoresis

  5. Add labelled DNA probes which hybridise / bind with target alleles (& wash to remove unbound probe)

  6. To show bound probe, expose membrane to UV light if a fluorescently labelled probe was used

  OR use autoradiography (expose to X-ray film) if a radioactive probe was used
• Explain how gel electrophoresis can be used to separate DNA fragments

  1. DNA samples loaded into wells in a porous gel and

  covered in buffer solution (which conducts electricity)

  2. Electrical current passed through → DNA is negatively

  charged so moves towards positive electrode

  3. Shorter DNA fragments travel faster so travel further

  
• What is a DNA probe? [2 marks]

  • Single-stranded DNA; ((Reject reference to a single strand of DNA))
  • Bases / sequence complementary to DNA / gene to be identified;
  • Radioactively / fluorescent) labelled so that it can be detected;