3.6 - Molecular biology toolkit

Question 1

recombinant

Answer 1

bringing together genetic material from different sources, including different species

Question 2

restriction enzymes

Answer 2

bacterial enzymes that cleave DNA in a highly specific manner

Question 3

how do restriction enzymes recognise cleavage sites

Answer 3

cleavage sites (4-8 nucleotides) that are palindromic and then cleave each strand of DNA

Question 4

agarose gel electrophoresis of DNA

Answer 4

1. DNA fragments generated by restriction enzyme
2. fragments visualised by staining with DNA-binding dye which fluoresces under UV light
3. DNA fragments loaded into well at cathode (-ve) end, fragments through gel towards anode (+ve) when voltage applied across gel
4. smaller fragments move quicker and travel further

Question 4

how can PCR be used for DNA fingerprinting? (2)

Answer 4

1. PCR capable of amplifying single molecule of DNA, can then be visualised in gel or sequenced
2. used to detect mutations that cause cancer, verify presence of infectious agents and determine guilt/innocence of crime suspects

Question 5

what does DNA fingerprinting make use of?

Answer 5

variability of non-coding “microsatellite” DNA between individuals

Question 6

plasmid

Answer 6

small, circular double-stranded DNA molecule separate from cell’s chromosomal DNA (found naturally in bacteria)

Question 7

insertion of DNA into plasmid vector (3)

Answer 7

1. restriction enzyme digest of DNA often generates fragments with staggered/”sticky” ends
2. vector can be cleaved by same restriction enzyme used to generate fragment, when fragment and cleaved vector mixed, sticky ends anneal
3. DNA ligase used to covalently join fragments and vector, making recombinant DNA

Question 8

reporter genes

Answer 8

reporter genes in vector, such as antibiotic resistance genes, make identification of vectors with inserter DNA sample easier

Question 9

expression of eukaryotic gene in E. coli (2)

Answer 9

1. expression plasmid drives transcription of cloned DNA insert to make large quantities within bacterial host cell
2. host cell translates mRNA to make protein, can be purified in large quantities

Question 10

what key human medicines are produced using expression of a eukaryotic gene in E. coli?

Answer 10

insulin - previously animal derived, harvested from cow/pig pancreas extracts

Question 11

how is a genomic library made

Answer 11

fragmenting entire genome of an organism and inserting fragments into a vector

Question 12

steps in constructing a genomic library (5)

Answer 12

1. genomic DNA cut into fragments of few thousand base pairs using restriction enzyme
2. fragments ligated (bound) into plasmid vector, plasmids transformed into E. coli bacterial host cells (each cell picks up single plasmid)
3. transformed E. coli propagated on nutrient plates
4. each bacterial colony division of single transformed E. coli cell (single clone)
5. aim is for every sequence in genome to be represented in total library of clones

Question 13

DNA sequencing

Answer 13

process of determining nucleic acid sequence (order of nucleotides in DNA)

Question 14

preparation of cDNA from eukaryotic DNA (4)

Answer 14

1. DNA copy of mRNA that is transcribed from active gees can also be made using RT-PCR
2. total mRNA extracted from selected type of cell (or embryo tissue etc) and DNA copy made using reverse transcriptase
3. RNA strand partially degraded using RNase, and complementary DNA (cDNA) strand synthesised using DNA polymerase
4. allows determination of genes expressed in certain cells, number of uses

Question 15

use of quantitive PCR (qPCR)

Answer 15

quantity of individual transcripts in cell can be determined by qPCR

Question 16

quantitive PCR (5)

Answer 16

1. mRNA isolated and converted into cDNA (complimentary DNA)
2. used as target DNA in PCR reaction carried out in presence of dye that fluoresces when bound to the duplex DNA
3. PCR cycle at which fluorescence becomes detectible is inversely related to original number of target templates
4. DNA sequence attached to solid support in defined pattern to generate microarray
5. fluorescently labelled cDNA then hybridised to microarray to reveal expression level of each gene on chip

Question 17

DNA microarrays

Answer 17

(gene chips) - allow determination of expression pattern of large number of genes simultaneously

Question 18

how can new genes be inserted into eukaryotic cells? (3)

Answer 18

1. calcium phosphate mediated uptake
2. microinjection into cells’
3. infection with retrovirus carrying gene of interest

Question 19

how can transgenic animals be generated?

Answer 19

some of DNA injected into embryos may be incorporates into the genome

Question 20

transgenic animals

Answer 20

useful tools for investigating a variety of diseases

Question 21

how can genes be knocked out?

Answer 21

genes can be disrupted (knocked out) when a foreign fragment is inserted by homologous recombination (genetic information between 2 similar sequences exchanged) - commonly used in DNA repair in cell

Question 22

how are transgenic mice produced? (4)

Answer 22

1. targeting vector used to disrupt sequence of specific gene in mouse embryonic stem (ES) cells
2. ES cells containing mutated gene injected into host embryos, then transferred to foster mother - allowed to develop to term
3. offspring derived from injection embryos are chimeras, contain cells from black host embryos and brown ES cells
4. different coat colours provide useful method of recognising chimeric offspring, then used for crossbreeding to produce fully transgenic mice

Question 23

CRISPR (2)

Answer 23

1. clustered regularly interspaced palindromic sequences
2. found in bacterial DNA

Question 24

genome editing using CRISPR-Cas9 (3)

Answer 24

1. Cas9 endonuclease directed to target by guide sequence and cleaves it
2. double strand break in target sequence can be repaired by non-homologous end joining, producing random insertions and deletions, inactivating target gene
3. more precise change to sequence can be introduced by providing ‘donor’ sequence for homologous recombination (HR)

Question 25

Cas proteins

Answer 25

near repeated clusters of genes encoding CRISPR-associated (i.e. Cas) proteins, function as “immune system” to bind and cleave foreign DNA sequences

Question 26

gene silencing by RNA interference (siRNAs)

Answer 26

1. double strand RNA (dsRNA) containing strand complementary to specific mRNA can be introduced into a cell
2. introduced RNA cleaved into small fragments, small interfering RNAs (siRNAs) by the enzyme dicer
3. complementary fragment to mRNA becomes incorporated into RNA-induced silencing complex
4. associated complementary RNA guides complex to mRNA which is then degraded
   - useful to study gene function

Question 27

what produces monoclonal antibodies?

Answer 27

immortal cell lines, identical antibodies produced by clones of single antibody producing cell

Question 28

how are immortal monoclonal antibody producing cells generated? (hybridoma cells)

Answer 28

generated by fusing normal, short-lived antibody-producing cells with immortal cells from a type of cancer called multiple myeloma - results in hybrid cells called hybridoma cells