L12 - Recombinant Technology

Question 1

What is the role of restriction enzymes?

Answer 1

Cut DNA into manageable size
Act as dimers and recognise short palindromic DNA sequence
Have precise recognition sequences

Question 2

Two types of restriction enzymes

Answer 2

Leave overhangs – sticky ends

Cut the DNA flush – blunt restriction enzymes

Question 3

How can restriction fragments be separated?

Answer 3

Can be separated by gel electrophoresis
DNA is negatively charged so is attracted to positive anode
Dyes such as ethidium bromide used to stain the DNA
DNA purified from a small slice of gel

Question 4

Cloning DNA - ligation method

Answer 4

Involves ligation of two DNA fragments to create recombinant DNA
Ligase hybridises sticky ends (cohesive termini)
- Recognition sequences are different but the overhangs are cohesive

Question 5

Cloning DNA - ligation of DNA fragments into plasmid method

Answer 5

Plasmid vectors are made by adding restriction enzymes sites in one part of the plasmid – multiple cloning site

Question 6

What are plasmids?

Answer 6

Small circular extra-chromosomal DNA
Occur naturally in bacteria 
Have their own origin of replication 
Usually results in 50 copies of the plasmid
Usually carry antibiotic resistant genes

Question 7

How much DNA do plasmid vectors hold?

Answer 7

<30 kilobases

Question 8

How much DNA do bacterial artificial chromosomes hold?

Answer 8

<300 kilobases

Question 9

How much DNA do yeast artificial chromosomes hold?

Answer 9

<3 megabases

Question 10

Cloning DNA - transformation of DNA into bacteria method

Answer 10

Involves mixing bacteria with the plasmid DNA and creating temporary holes in the cell membrane

• Electroporation or chemical treatment
• Competent bacteria – bacteria that are ready to take up new DNA
• Not very efficient process so treated bacteria are selected on antibiotic plates

Colonies contains thousands of bacteria that each contain 50 plasmids originating from single plasmid
- Single colonies are lifted from plate to start a liquid culture

Question 11

Library of genomic advantages

Answer 11

Contains all the regulatory sequences

Allows one to study transcriptional regulation

Question 12

Library of cDNA clones from mRNA advantages

Answer 12

Only contains genes that are expressed
Studying disease involves identify genes expressed in diseased tissue
- Help identify genes involved with the disease

Question 13

Cloning cDNA method

Answer 13

1. Extract RNA from cancer tissue
2. Reverse transcription reaction on mRNA
3. Ligate new DNA copy into a plasmid vector and transform into bacteria
4. Grow bacterial culture and purify the clone

cDNA will represent genes that are expressed in the original tissues – the transcriptome

Question 14

cDNA library method

Answer 14

1. Cancer tissue
2. Population of RNA reverse transcribed into DNA
3. Purify clones

Genomic labs sequence the ends of all the clones in the library – expressed sequence tags

Question 15

To isolate single clones from a mixed population it is important there is only

Answer 15

One insert in each plasmid
One plasmid in each bacteria
One bacteria starts each colony

Question 16

When cloning gene some clones will be

Answer 16

• House keeping genes
• Tissue specific

Highly transcribed genes will be cloned more often
More interested in regulatory genes that are rare

Question 17

Genomic library method

Answer 17

1. Human cell
2. Purify and digest chromosomal DNA with restriction enzymes
3. Purify 1000s of different clones

Question 18

Dideoxy terminator sequencing method

Answer 18

1. Denature template so its single stranded
2. Allow to cool with the primer
3. Start DNA synthesis reaction with DNA polymerase and dNTPs
4. DNA polymerase cannot extend a strand once a ddNTP has been added
5. If we add mix of deoxy and dideoxy nucleotides – different strands will end at different positions

Running all four ddNTP reactions on the same gel results in a nucleotide ladder

Question 19

What are primers?

Answer 19

Short (20 nt) single stranded DNAs that can easily be synthesised
Designed to anneal to sequence on edge of vector

Question 20

Automated sequencing method

Answer 20

1. Rather than labelling primer - ddNTPs are labelled with a flurescent dye
2. While sequencing gel is running a camera takes pictures of each band and measures its intensity
   o The camera takes a picture at one position over time
3. Results presented as a graph showing intensity over time – trace

Question 21

When are progressive and shot gun sequencing used?

Answer 21

When the DNA is larger than 1 kb

Question 22

Progressive sequencing method

Answer 22

Ends of clones are sequenced using primers from the vector
Primers designed based upon the new sequence
More rounds of sequencing are performed until sequences meet in the middle
BAC clones - localized to particular regions of genome and chosen for sequencing

Question 23

Shot gun sequencing method

Answer 23

Make genomic plasmid library and sequence the ends from each clone using primers
Short random sequences (traces) are assembled by a computer program into a contig to see if they overlap

Question 24

Shot gun sequencing advantages

Answer 24

Automated

Question 25

Shot gun sequencing disadvantages

Answer 25

Need to sequence 6X the size of the genome to get large contigs
Inefficient - will always be gaps

Question 26

Both progressive and shot gun sequencing are usually used in parallel

Answer 26

Human genome has 3.29 DNA base pairs containing 23,000 genes
1990 - 2003 - cost many millions
With new technology 1 machine can sequence a genome in 56 hours