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19.1.919 · Genetic Technology

Gel Electrophoresis — Cambridge AS & A Level Biology 9700 Revision Notes & Topical Questions

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📚 Cambridge AS & A Level Biology 9700 Hub / Topical Revision
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19.1.9 Gel Electrophoresis — CAMBRIDGE AS & A LEVEL BIOLOGY 9700 — REVISION NOTES notes: Explain how gel electrophoresis separates DNA fragments and proteins and how it is used in DNA profiling.Gel electrophoresis — widely used to analyse DNA, RNA and proteins by separating molec…

  • Limitations
  • Polyacrylamide
  • DNA is negatively charged because of its phosphate groups, so it moves towards the anode
  • Tiny pores let smaller molecules move quickly and larger ones slowly
  • Different gels have different pore sizes
  • DNA can be collected from almost any body tissue, e.g. hair root or saliva

Section 1 of 9Principle

Gel electrophoresis
  • Gel electrophoresis widely used to analyse DNA, RNA and proteins by separating molecules according to size and net charge.
Positively charged molecules move to the cathode (negative pole); negatively charged molecules move to the anode (positive pole).
DNA is negatively charged because of its phosphate groups, so it moves towards the anode.
Molecules move through the gel at different rates.
  • Agarose gel is used for DNA and polyacrylamide (PAG) for proteins.
  • Tiny pores let smaller molecules move quickly and larger ones slowly.
  • Different gels have different pore sizes.

Section 2 of 9Preparing DNA

Preparing DNA
DNA can be collected from almost any body tissue, e.g. hair root or saliva.
The DNA is first amplified by PCR.
Restriction endonucleases cut it into fragments near variable number tandem repeats (VNTRs).
  • VNTRs are non-coding regions with variable numbers of repeats, called satellite or microsatellite DNA.
  • They vary between people except identical twins.

Section 3 of 9Separating DNA fragments

1
Pour liquid agarose into a tank and let it set as a gel.
2
Use a comb to mould wells at one end of the gel.
3
Submerge the gel in an electrolyte solution.
4
Load the fragments into the wells with a micropipette.
5
Apply an electric current, with the negative electrode at the wells end.
6
The negatively charged fragments move towards the anode; shorter fragments travel faster and further.
7
Transfer the fragments to absorbent paper or nitrocellulose, which is heated to separate the strands.
8
Add probes, then take an X-ray image or shine UV light to reveal the bands, comparing with a control.

Section 4 of 9Probes

Probes
  • Probes single-stranded DNA sequences complementary to the VNTR regions being sought.
A radioactive label (e.g. a phosphorus isotope) darkens X-ray film to show bands.
A fluorescent dye fluoresces under UV light to show coloured bands.
Bands are compared with a DNA ladder of known sizes to estimate fragment size.

Section 5 of 9Separating proteins

Separating proteins
The amino acid composition determines the charge on each protein, because R groups carry different charges.
The charge of R groups depends on pH, so buffer solutions keep the pH constant.
Electrophoresis separates polypeptides made by different alleles, e.g. haemoglobin variants including the sickle cell β-globin.

Section 6 of 9Uses and limits

DNA profiling (genetic fingerprinting) for forensic work and paternity.
Genetic screening, e.g. different alleles give different fragment patterns.
Checking PCR and restriction digest products in genetic engineering.
Limitations
  • Limitations gives only approximate sizes, poor resolution of similar sizes, and contamination gives false bands.
Some stains are mutagenic and UV light is harmful, so safe handling is needed.

Section 7 of 9More from the specification

1Isolation of Desired Genes
  • Scientists isolate specific target genes from cells using restriction enzymes or by synthesising cDNA from extracted mRNA templates using the enzyme reverse transcriptase.
2Polymerase Chain Reaction (PCR)
  • PCR employs heat-stable DNA polymerase and specific primers to amplify minute quantities of target gene sequences through automated cycles of denaturation, annealing,
  • and extension.
3Bacterial Transformation and Vectors
  • Desired genes are inserted into bacterial plasmids using DNA ligase,
  • and the recombinant vectors are introduced into host bacteria cells to express foreign proteins.
4Isolation of Desired Genes
  • Scientists isolate specific target genes from cells using restriction enzymes or by synthesising cDNA from extracted mRNA templates using the enzyme reverse transcriptase.
5Polymerase Chain Reaction (PCR)
  • PCR employs heat-stable DNA polymerase and specific primers to amplify minute quantities of target gene sequences through automated cycles of denaturation, annealing,
  • and extension.
6Bacterial Transformation and Vectors
  • Desired genes are inserted into bacterial plasmids using DNA ligase,
  • and the recombinant vectors are introduced into host bacteria cells to express foreign proteins.
7Promoter
  • DNA region where RNA polymerase binds to start transcription.
8Genome
  • All the genetic information in an organism.
9Nucleotide
  • The unit of DNA made of a phosphate, a deoxyribose sugar and a base.
10Reverse transcriptase
  • Enzyme synthesising DNA from an RNA template
11Marker gene
  • Gene coding for a detectable substance, used to identify modified cells.
12Genetically modified
  • Having had genes from another organism inserted.
13Organism
  • A living thing, such as a plant or an animal.
14Bacteria
  • Single-celled microorganisms, much smaller than plant or animal cells.
15Enzyme
  • A protein that functions as a biological catalyst.
16Specific
  • Able to act on only one type of substrate.
17Plasmids
  • Convenient vectors
18Plasmid
  • A small ring of DNA in a bacterium carrying extra genes.
19Recombinant
  • Offspring with a new combination of characteristics formed by crossing over.
20Microarray
  • Chip with probes used to detect many genes at once.

Section 8 of 9Key terms

TermMeaning
Gel electrophoresisSeparation of molecules by size and charge in a gel in an electric field.
AgaroseGel used to separate DNA.
VNTRVariable number tandem repeat: non-coding repeated sequence varying between people.
ProbeLabelled single-stranded DNA complementary to a target sequence.
AnodeThe positive electrode.
PolyacrylamideGel used to separate proteins.

Section 9 of 9Exam technique

1
Saying DNA moves to the negative pole — it is negatively charged and moves to the positive pole (anode).
2
Saying larger fragments move further — smaller fragments travel faster and further.
3
Placing the wells at the positive end — wells are at the negative end.
4
Forgetting that DNA must be cut into fragments before electrophoresis.

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