Genetics 7: Gene library

Question 1

What is recombinant DNA technology

Answer 1

Genes or DNA from two different sources combined in vitro in the same molecule.

Question 2

What is the role of restriction enzymes in recombinant DNA technology

Answer 2

Restriction enzymes are isolated from bacteria (originally protection for bacteria against bacteriophages)
They cleave the sugar phosphate backone at specific symmetrical sequences 4-8bp long called restriction sites in a staggered way called sticky ends.

Question 3

What is the role of DNA ligase in recombinant DNA technology

Answer 3

DNA ligase catalyses the formation of covalent bonds that close the sugar phosphate backbone between insert DNA and cloning vector together

Question 4

Why are DNA insert and cloning vector cut using the same restriction enzyme

Answer 4

They produce sticky ends on each DNA piece that are complementary, allowing the two pieces to H bond together

Question 5

What is a plasmid

Answer 5

Self replicating circular autonomous piece of DNA molecule which can replicate inside host bacterial cells.
Can have more than one

Question 6

What are the steps of cloning a single gene into a plasmid

Answer 6

1. Plasmid has an antibiotic resistant gene already and a ‘lac Z’gene in the middle of the restriction site which codes for enzyme cleaving blue dye.
2. The plasma extracted from bacteria and cut with restriction enzyme
3. DNA from donor purified, cut with same restriction enzyme.
4. Combined together to make recombinant plasmid + some non recombinant plasmid that closed on itself
5. Electric shock or chemical holes on each bacteria to take up one plasmid.
6. Bacteria colony is grown and those without plasmid killed off by antibiotic.
   Those that have non recombinant plasmid still have intact LacZ gene so can cleave blue dye

Question 7

Where are restriction sites usually in a plasmid

Answer 7

multiple cloning site of plasmid, driven by a promoter.

Question 8

What is a genomic library compared to cDNA library

Answer 8

Genomic library : A collection of clones of bacteria that together contain an organisms entire genome= nuclear, mitochondrial + repeat sequences, promoters in introns.

CDNA library: A collection of clones containing all of the gene sequences that are expressed in a particular tissue at time of RNA extraction. Less complex as it doesn’t have promoters, UTR and introns.

Question 9

What are the two types of genomic libraries and what method of cloning is used to produce them

Answer 9

The library is made by cloning a mixture of small fragments representing the entire genome = shotgun cloning.

Plasmid library uses smaller fragments so need a lot of plasmid clones.
BAC (Bacterial artificial chromosome) has larger inserts so doesn’t need as many can be stored on multiwelled plates

Question 10

How is cDNA library made

Answer 10

1. The nucleus creating the mRNA in the cytoplasm is isolated
2. A poly T primer is made for Poly A tail of mRNA
3. Reverse transcriptase can be used to make DNA from mRNA. This is a single strand DNA copy.
4. Another enzyme digests away the RNA and use a primer to get DNA polymerase to make the final strand.

Question 11

How are libraries screened (finding a gene of interest) by hybridisation

Answer 11

1. Double stranded target DNA is separated into single strands by heating/ chemicals on a gridded library
2. DNA probe = single stranded DNA fragment with similar sequence to the gene of interest is washed over the sequences.
3. DNA probe will complementary pair to the sequence.
4. The radioactive/fluorescent tag is used to pick up the DNA and wash the other DNA out.

Question 12

What reaction makes copies of DNA made and what are the reagents of this reactions

Answer 12

PCR reaction uses

1. Heat stable DNA polymerase, 2.deoxyribonucleotides
2. Two primers (one for each strand as DNA polymerase can only extend existing double stranded regions.

Question 13

What are the steps for PCR reaction

Answer 13

1. Heat the sample up to 90 to separate the two strands of DNA
2. Cool the mixture to 50 to allow primers to anneal to single strands
3. Heat up to 70 to allow TAC polymerase to extend the sequence out and produce 2 copies of the DNA

Question 14

How does Gel electrophesis work and what is its purpose

Answer 14

Purpose: determine size of DNA strands by comparing rate of movement of negatively charged DNA fragments through an agarose gel in an electric field (from cathode to anode). This is compared to a DNA ladder.
The gel is run over a fixed amount of time and uses a dye to visualise the segments.

Question 15

What does the rate of movement of a macromolecule in gel electrophoresis depend on

Answer 15

The size of fragment, electrical charge and other physical properties of macromolecule.

Question 16

What is the purpose of Capillary DNA sequencing and how does it work

Answer 16

Purpose: To determine nucleotide sequence
Works:
One strand is used to synthesis a nested set of complementary fragments. Synthesis is terminated randomly by the addition of a fluorescently tagged dideoxyribonucleotide (ddNTP). Each nucleotide has a different fluorescent tag.
So when these are random fragments are gel electrophoresed they are separated by size and the total sequence can be read.

Question 17

Compare a BAC library and a Plasmid library

Answer 17

Plasmid library uses smaller restriction indentification so has smaller fragments
whereas BAC can use 100-300 kb fragments as bigger restriction identification