Molecular techniques 2

Question 1

PCR AMPLIFIES SELECTED REGIONS OF DNA in vitro.. explain how

Answer 1

a. Region of target DNA to be amplified

1. Add oligonucleotide primers
2. Heat to separate strands (95 C)
3. Cool; primers ANNEAL (55 - 65 C)
4. hEAT TTO 72 C to allow DNA synthesis.

If know sequence of parts of gene can amplify in a test tube.

Use multiple copies of a pair of short, chemically synthesised
primers– bind eachDNA strand (3’ ends point at each other).

5. REPEAT STEPS 2,3 AND 4
   After 25 cycles, the target sequences has been amplified about 10^6-fold
6. Polymerases add nts to primers
7. Polymerisation shuttles back and forth forming exponentially growing number of specific dsDNA.

Question 2

PRODUCING PCR PRODUCTS with Sticky ends…

Answer 2

1. Initial steps of PCR (heats to 95 C, then cool to anneal and synthesise DNA)
2. Second round of PCR
3. Further rounds of PCR
4. Digest with EcoRI
5. DNA that arise from regular PCR have BLUNT or near blunt ends ( depends on polymerase used - TA cloning). While blunt end vector with ligase, very INEFFICIENT.
6. Create PCR products with sticky ends- use designed PCR primers containing REs sites.
7. Digestion of final PCR product with ER produces fragment ready to be inserted into a vector.

Question 3

DNA copies of mRNA can also be synthesised:

Answer 3

1. to analyse genes that encode proteins, mRNA is better predictor of polypeptide sequence than genomic sequence.
2. Complementary DNA (cDNA) = a DNA version of an mRNA molecule (more stable)
3. cDNA made by an enzyme called Reverse transcriptase. Must isolate RNA from a tissue in which the gene is expressed.
4. Intron EXon, gene seg.
   - Transcription (in cell)
   - Introns removed (in cell)
   - OligodT primer anneal tp poly A tail
   - Reverse transcriptase
   - mRNA
   - Single-stranded cDNA
   - DNA polymerase copies
   - cDNA
   - 3’ Double-stranded
   - 5’ cDNA

NOTE: When mRNA strand has degraded (RNAseH), addition of second primer
(Not shown) premits initiation by DNA polymerase, completes synthesis.

Question 4

Producing cDNA molecules with sticky ends…

Answer 4

• Double stranded cDNA
• Ligate oligonucleotide
  -linkers containing
• EcoRI sequence

Cut with EcoRI

Ligate into vector cut with EcoRI

Adding EcoRI sites to end of cDNA molecule.
Linkers or adaptors (boxed) added to both ends of cDNA molecule.

Question 5

CLONING GENES:

Answer 5

One way to amplify a specific piece of DNA is to place fragment into bacterial cell.

termed “gene cloning” as identical copies (clones) of original DNA produced.

Question 6

A cloning vector is a stable, replicating DNA molecule foreign DNA can be attached 3 important features:

Answer 6

1. Origin of replication (allows DNA replication)
2. Selectable markers (e.g. antibiotic resistance)
3. Unique restriction enzyme sites into which DNA fragment inserted (MCS = multiple cloning site)

Question 7

PLASMID VECTORS:

Answer 7

Circular DNA molecules exist naturally in bacteria

Origin of replication, so can replicate independently of bacterial chromosome.

Ones used in cloning specially constructed to contain multiple RE sites.

Question 8

What should plasmid vector s have?

Answer 8

1. Origin of replication recognised in the host cell so that replicated along with the DNA that it carries.
2. Should carry selectable markers - traits that enable cells containing the vector to be selected or identified.
3. Single cleavage site for each of 1 or more restriction enzyme used.

Question 9

Method for inserting foreign DNA into plasmid vector:

TO CREATE RECOMBINANT DNA

Answer 9

1. Both DONOR AND VECTOR DNAs digested by a RE that produces complementary sticky ends
2. Resulting fragments are mixed in test tube to allow sticky ends to hybridise.

Question 10

Amplification of donor DNA inside a Bacterial cell:

Answer 10

A single recombinant vector enters bacterial cells and is amplified by replication machinery -> MANY COPIES OF EACH VECTOR IN BACTERIA

After amplification, a colony of bacteria will contain billions of copies of donor DNA insert fused to vector (CLONE)

AMPLIFICATION STEPS:
- choose cloning vector and insert DNA of interest
- introduce recombinant DNA to bacteria
- Recover amplified recombinant molecule.

Question 11

CHOICE OF CLONING VECTOR - numerous vectors suitable for different size inserts

Answer 11

Plasmid - Size of DNA that can be cloned: As large 15 kb. Method propagation: Plasmid replication, INTRODUCTION TO BACTERIA: TRANSFORMATION.

Phage lambda - as large as 23 kb, Phage reproduction, phage infection.

Cosmid, 44 kb, plasmid reproduction, phage infection.

Bacterial artificial chromosome..300kb, plasmid reproduction, electrporation.

Question 12

PLASMID VECTORS

Answer 12

1. Small, circular DS DNA replicate INDEPENDENTLY of bacterial chromosome.
2. Plasmid vectors usually contain a gene for drug resistance (eg ampR) and a Gene to distinguish plasmid with and without insert ( eg lacZ)

Multiple cloning site.

Question 13

Understanding Bacteriophage Vectors
;

Answer 13

1. Harbours DNA as insert “packaged” inside phage particle.

In lambda - the nonessential central region of phage chromosome discarded

Ends ligated to random 15kb (can be up to 23kb) fragments of donor DNA.

Linear multimer forms, then stuffed into phage heads as monomer.

Question 14

FOSMIDS (type of cosmid vector) and BACs are cloning vectors that carry large inserts

Answer 14

• Vectors that carry 35kb to 45kb inserts
• engineered hybrids of lambda phage DNA and bacterial plasmid.
• packaged into Lambda phage, which act as syringe and introduce DNA into bacteria
• when in cell, these hybrids form circular molecules, replicate extrachromosomally (like plasmids)
• very few copies accumulate in cell

Question 15

BACs: Bacterial artificial chromosomes = 3

Answer 15

1. most popular cloning vector for large DNA inserts
2. Derived from F plasmid (controls mating/transfer genetics material), cam carry 100-200kb inserts, vector itself only 7kb.
3. Large circular recombinant DNA introduced into bacteria.

Question 16

YACs: Yeast Artificial Chromosomes

Answer 16

1. • DNA molecules that has yeast origin replication, a pair of telomeres and a centromere
2. • stable, replicate, segregate as yeast chromosomes.
3. carry 600kb inserts ( up to 1000kb)

Question 17

Modes of delivery recombinant DNA into bacteria : PLASMIDS, BACs….FOSMIDS

Answer 17

PLASMIDS, BACS
- Transformation: bacteria bathed in solution containing recombinant DNA. Bacteria unable to take up large plasmids, made “competent” DNA enters competent cell through membrane pores, recombinant molecule becomes plasmid.

Electroporation of BACs.

FOSMIDS
TRANSDUCTION: recombinant molecule combined with phage head and tail proteins. Mixed with bacteria, inject DNA cargo into bacterial cells. FOSMIDS.

Question 18

MODES OF DELIVERING RECOMBINANT DNA INTO BACTERIA:

BACTERIOPHAGE VECTORS

Answer 18

INFECTION: produces recombinant phage particles.
Repeated rounds of re-infection, plaque full of phage particles forms from each bacterium infected.

Each phage particle contains copy of recombinant lambda chromosome.

RECOVERY: Collected phage lystate, isolate DNA or BREAK OPEN BACTERIA (plasmids, fosmids)

Question 19

What is a DNA LIBRARY?

Answer 19

A collection of clones containing all the DNA fragments from one source.

eg. Isolate genomic DNA from human cells, fragment, clone ALL into bacterial cells/phage.

Question 20

Genomic DNA library?

Answer 20

Contains all the DNA sequences in the genome

Question 21

cDNA library

Answer 21

consisting only of those DNA sequences that are transcribed into mRNA.

Question 22

Genomic libraries contain all the DNA sequences found in an organism’s genome

Answer 22

CELLS COLLECTED, DISRUPTED, DNA EXTRACTED
RE added for a limited TIME ONLY (PARTIAL DIGEST) SO THAT ONLY SOME OF THE RE SITES ARE CUT

1. Multiple copies of genomic DNA are digested by a restriction enzyme for a limited time so that only some of the restriction sites in each molecule are cut.
2. Different DNA molecules are cut in different places, providing a set of overlapping fragments
3. Each fragment is then joined to a cloning vector
4. and Transferred to a bacterial cell,…
5. producing a set of clones containing overlapping genomic fragments, some of which may include segments of the gene of interest.

CONCLUSION: some clones contain the entire gene of interest, others include part of the gene, and most contain none of the gene of interest.

A GENOMIC LIBRARY MUST CONTAIN A LARGE NUMBER OF CLONES TO ENSURE ALL DNA SEQUENCES IN GENOME ARE REPRESENTED.

LIBRARY OF HUMAN GENOME USING COSMIDS (35-35kb) REQUIRE APPROX 350 000 CLONES TO PROVIDE 99% CHANCE EVERY SEQUENCE INCLUDED IN LIBRARY.