L7: Molecular Bio Techniques I

Question 1

What does recombinant DNA technology allow?

Answer 1

Allow isolation & manipulation of DNA

Question 2

How is a recombinant DNA molecule created?

Answer 2

Isolated DNA fragments are inserted into a cloning vector

Question 3

What is a cloning vector?

Answer 3

DNA molecule that can be introduced into a host organism & can self-replicate

Question 4

What are restriction enzymes?

Answer 4

Function as ‘molecular scissors’ to cut DNA

Question 5

How do restriction enzymes function?

Answer 5

Recognised specific nucleotide sequences in the DNA & cut both strands of the sugar-phosphate backbone

Question 6

Define palindrome

Answer 6

Sequence that is read the same both ends

Question 7

What do restriction enzymes leave when they are cut in a cleavage?

Answer 7

Sticky/blunt ends

Question 8

What does the number & size of fragments of DNA depend on?

Answer 8

• Restriction enzyme
• Size of genome
• Abundance of each nucleotide

Question 9

How are restriction maps created?

Answer 9

Cutting DNA with individual restriction enzymes

Question 10

How is the digested DNA separated using?

Answer 10

Agarose gel electrophoresis which separates DNA frgaments by size

Question 11

How are the DNA fragments size identified?

Answer 11

Comparing how far they move in the gel compared to DNA fragments of known sizes

Question 12

What is bacteria/yeast used in recombinant DNA?

Answer 12

Replicate & amplify individual DNA fragments

Question 13

What is the use of plasmids in recombinant DNA?

Answer 13

Used as ‘cloning vectors’ to carry DNA fragments to host

Question 14

What do plasmids contain?

Answer 14

Antibiotic (AB) resistance genes allowing selective growth of bacteria containing plasmids

Question 15

What is the role of plasmids

Answer 15

Carry gene for replicating their DNA

Question 16

What are plasmids used for?

Answer 16

DNA cloning

Question 17

2 different types of plasmids

Answer 17

1) Cloning plasmids for cloning genes
2) Expression plasmids adapted to allow gene expression

Question 18

How can a DNA molecule be cloned ?

Answer 18

Inserting it into a plasmid cloning vector

Question 19

How is a recombinant DNA molecule created?

Answer 19

DNA cut by same restriction enzyme will have same cohesive ends with complementry base pairs, these will anneal to one another

Question 20

How is a DNA fragment cloned?

Answer 20

1) DNA fragment & cloning vector (e.g plasmid) cut with the same restriction enzyme

2) They have the same complementary cohesive ends

3) Cut DNA fragments & vector are mixed together

4) DNA fragments will anneal to cohesive ends in vector

5) DNA ligase ligate the DNA fragment to form recombinant DNA molecule

Question 21

Where is cloned DNA amplified?

Answer 21

E.coli

Question 22

How is cloned DNA amplified?

Answer 22

Individual recombinant plasmids is taken up by E.coli & using the origin of replication (ori), 100-200 copies of it is generated

Question 23

What does lacZ encode?

Answer 23

Encodes beta-galactosidase which acts on X-gal to produce blue bacteria

Question 24

Describe genomic DNA library

Answer 24

1) Human DNA cut with restriction nuclease to create millions of DNA fragments

2) DNA fragments inserted into plasmids using ligase to create recombinant DNA molecules

3) Plasmids is then introduced into bacteria to create a genomic library

Question 25

Uses of cloned DNA

Answer 25

1) Identify changes in the genome 2) Characterise how genome is organised 3) Engineer organisms by transferring genes between organisms

Question 26

Why can't genomic DNA be used for studying/use in therapies?

Answer 26

Contains introns ## Footnote Isolate mRNA where introns are removed to create cDNA

Question 27

How is complementary DNA (cDNA) made?

Answer 27

DNA copy of a mRNA, using an enzyme reverse transcriptase

Question 28

What do cDNA libraries contain?

Answer 28

Complementary DNA copies of the mRNA present in a cell population & represent genes being expressed in a population

Question 29

Where is cDNA used?

Answer 29

Used in eukaryotic expression vectors as introns are removed

Question 30

Does mRNA contain introns?

Answer 30

NO

Question 31

What is a cDNA library?

Answer 31

Large collection of plasmids each containing a single cDNA

Question 32

What do frequencies in genomic DNA & cDNA only include?

Answer 32

Only sequences found in mature mRNA

Question 33

What are expression plasmids?

Answer 33

Plasmid cloning vectors that allow expression of cloned genes in bacteria

Question 34

What are eukaryotic expression plasmids?

Answer 34

Plasmid cloning vectors that allow expresison of cloned genes in eukaryotic tissue culture cells

Question 35

What is cloning vector and expression vector?

Answer 35

Cloning vector is a small piece of DNA maintained in bacteria Expression plasmid used to introduce cloned DNA into bacteria to allow expression of cloned gene

Question 36

# Cloning vector vs Expression vector Uses

Answer 36

Cloning vector used to introduced cloned DNA into bacteria to make lots of copies of insert DNA Expression vector used to obtain gene produce of cloned DNA (protein/RNA)

Question 37

# - What does nucleic acid hybridisation allow?

Answer 37

Allow identification of nucleic acid fragments/clones containing specific sequences - Relies on comlpementarity of strands of nucleic acid

Question 38

Explain the nucleic acid hybridisation principle

Answer 38

Individual strands of DNA separated by heating - After cooling the complementary strands anneal together which 'hybridise' one another

Question 39

Explain nucleic acid hybridisation technique

Answer 39

Used to identify DNA/RNA that match a specific sequence using ssDNA as a probe to identify fragments in a collection that have a sequence complementary to the probe DNA

Question 40

Define probe

Answer 40

Single-stranded sequence of DNA/RNA used to identify specific sequences of DNA/RNA

Question 41

What is the DNA probe labelled with?

Answer 41

Using DNA polymerase to incorportate labelled dNTPs

Question 42

How can dNTPs be labelled?

Answer 42

Making them radioactive using 32P OR Attaching a fluorescent molecule OR Attaching digoxygenin (STEROID MOLECULE)

Question 43

What is nucleic acid hybridisation used to?

Answer 43

To identify DNA fragments in the genome that contain specific genes

Question 44

What is used in the southern blot?

Answer 44

DNA fragments separated by gel electrophoresis

Question 45

Use of genomic southern blot

Answer 45

Allow identification of genomic DNA fragments generated by restriction enzymes containing a specific DNA sequence

Question 46

Use of a clone blot

Answer 46

Allow identification of restriction fragments in cloned DNA that contain DNA sequence matching probe DNA