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ESA1 - Medical Cell Biology and Genetics > Introduction to Molecular Techniques > Flashcards

Introduction to Molecular Techniques Flashcards

1
Q

What techniques are involved in analysis of DNA at the gene level?

A

Restriction enzymes, DNA gel electrophoresis and PCR

Also southern hybridisation, microarray, PCR variations

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2
Q

What techniques are involved in analysis of protein?

A

Protein electrophoresis, immunoassays and enzyme assays

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3
Q

What technique is involved in analysis of DNA at the nucleotide level?

A

DNA sequencing

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4
Q

What techniques are involved in analysis of DNA at the chromosome level?

A

Karyotyping and FISH

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5
Q

What are restriction enzymes?

A

Enzymes that cut a DNA molecule at a particular place

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6
Q

What bonds do restriction enzymes cut?

A

Phosphodiester

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7
Q

What do restriction enzymes recognise and cut?

A

Specific DNA sequences (restriction sites) - mostly palindromic

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8
Q

What restriction site does EcoRI recognise?

A

GAATTC

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9
Q

What does EcoRI digestion produce?

A

Sticky ends (complementary base pairs)

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10
Q

How does DNA gel electrophoresis separate DNA fragments?

A

Based on their size - DNA is negatively charged due to the phosphate groups so they will move towards the positive electrode. Smaller fragments move further.

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11
Q

What are the requirements for gel electrophoresis?

A
  1. Gel - matrix allowing separation of fragments
  2. Buffer - allows charge on the DNA samples across gel
  3. Power supply - generate charge difference
  4. Stain/detection - identify presence of separated DNA (fluorescent markers)
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12
Q

Why would we use restriction analysis?

A
  • To investigate size of DNA fragments to see if there are any changes in DNA (eg small deletions)
  • To investigate mutations (eg sickle cell disease)
  • To investigate DNA variation (DNA fingerprinting)
  • To clone DNA
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13
Q

What are the four basic steps of gene cloning?

A
  1. Isolate relevant gene following digestion with restriction enzymes
  2. Insert gene of interest into plasmid vector (recombinant DNA molecule)
  3. Introduce recombinant DNA molecule into suitable host cells (eg Ecoli)
  4. Identidy and isolate the clone containing the DNA of interest
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14
Q

Why would we clone human genes?

A

To make useful proteins eg insulin

To find out what genes do eg HTT

Genetic screening eg Huntingtons

Gene therapy

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15
Q

What does reverse transcriptase do?

A

Converts RNA to cDNA

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16
Q

What is the enzyme used in the Polymerase Chain Reaction?

A

Taq polymerase

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17
Q

What does PCR require?

A

Taq polymerase
Primers
Nucleotides

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18
Q

Why is taq polymerase used in PCR/

A

It is thermostable

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19
Q

What is the purpose of PCR?

A

To amplify DNA

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20
Q

What are the basic steps of PCR?

A
  1. At temp of 90 degrees, DNA denatures and hydrogen bonds are broken
  2. At temp of 60 degrees, primers anneal
  3. At temp of 72 degrees, TAQ polymerase adds nucleotides to complementary strands
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21
Q

In PCR, what defines the region to be copied?

A

Pair of primers - forward and reverse

22
Q

Why would we use PCR?

A

To amplify a specific DNA fragment

Investigate single base mutations

Investigate small deletions or insertions

Investigate variation and genetic relationships

23
Q

How can proteins be separated in gel electrophoresis?

A

Basis of size, shape or charge

24
Q

What technique is used for the separation of proteins on the basis of size?

A

SDS-PAGE (use of detergent)

25
Q

What technique is used for the separation of proteins on the basis of charge?

A

Isoelecric focusing (IEF)

Proteins migrate until they reach a pH equal to their pI (no net charge at pI so stop migrating)

26
Q

What technique allows the separation of a complex mixture of proteins?

A

2D-PAGE (important for diagnosing disease states in different tissues)

Firstly separate on basis of pH, then as there may be multiple proteins on each band, separate on basis of size

27
Q

How are proteins identified?

A
  1. Digest protein with trypsin
  2. Perform mass spectrometry
  3. Generate list of peptide sizes
  4. Use database of predicted peptide sizes for known proteins to identify the protein
28
Q

What amino acids are cleaved by trypsin?

A

Lysine, Argenine

29
Q

What amino acids are cleaved by staphylococcal protease?

A

Aspartate, glutamate

30
Q

What amino acids are cleaved by chymotrypsin?

A

Tyr, Trp, Phe, Leu

31
Q

What amino acids are cleaved by endopeptidase Lys-C?

A

Lysine

32
Q

What does hydroxylamine cleave?

A

Asp-Gly bonds

33
Q

What does cyanogen bromide cleave?

A

Methionine

34
Q

What does western blotting detect?

A

Specific proteins

35
Q

What technique can be used to measure the concentration of proteins in solution eg hormones?

A

Enzyme-linked immunoabsorbent assay (ELISA)

36
Q

What assay method is used in the measurement of the concentration of cortisol?

A

RIA (radioimmunoassay)

37
Q

What assay method is used in the measurement of the concentration of insulin?

A

RIA, ELISA

38
Q

What assay method is used in the measurement of the concentration of TSH?

A

RIA, ELISA

39
Q

What assay method is used in the measurement of the concentration of T3 and T4?

A

RIA, ELISA

40
Q

What condition is cortisol increased in?

A

Cushing disease

41
Q

What condition is cortisol decreased in?

A

Addison disease

42
Q

What condition is insulin increased in?

A

Obesity

43
Q

What condition is insulin decreased in?

A

Type I diabetes

44
Q

What condition is TSH increased in?

A

Hypothyroidism

45
Q

What condition is T3/T4 increased in?

A

Hyperthyroidism

46
Q

Why does SDS-PAGE separate on basis of size?

A

SDS disrupts tertiary structure of proteins to bring the folded proteins down to linear molecules. It also coats the proteins with uniform negative charge

47
Q

Why does SDS-PAGE separate on basis of size?

A

SDS disrupts tertiary structure of proteins to bring the folded proteins down to linear molecules. It also coats the proteins with uniform negative charge

48
Q

How can glucose concentration be measured?

A

Glucose oxidase

Test strips - H2O2 converted to coloured dye

49
Q

What enzymes are markers for liver damage/disease?

A

Aspartate transaminase and Alanine transasminase (AST and ALT)

gamma-glutamyl transferase - increased by alcohol

50
Q

What enzymes are markers for pancreatitis?

A

Amylase/lipase

51
Q

What enzyme is a marker for bone disorders?

A

Alkaline phosphatase

ESA1 - Medical Cell Biology and Genetics (18 decks)

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