rDNA technology and genomics

Question 1

How can DNA be prepared for studying?

Answer 1

• homogenise cells or tissues
• centrifuge.. the aqueous layer is nucleic acids and the phenol layer is protein
• addition o ethanol and salt causes the nucleic acids to precipitate out of solution

Question 2

How are type II restriction endonucleases used in DNA extraction?

Answer 2

• cut from within the DNA to cleave specific sequences
• usually cleave palindrome sequences
• cuts both strands of DNA causing them to separate
• the sticky ends are then left unpaired with DNA (overhang)

Question 3

How can compatible DNA overhangs be re-joined?

Answer 3

When the DNA is cleaved it can produce compatible overhangs that when combined are no longer recognised by the endonuclease.
When they are rejoined a gap remains that is rejoined by DNA ligase

Question 4

What is agarose gel electrophoresis?

Answer 4

• separates DNA fragments by size and charge
• DNA moves towards a positive electrode at varying speeds based on its size
• its exact size is then determined using a mwt marker made of fragments of known sites

Question 5

What is recombinant DNA?

Answer 5

an artificial DNA sequence generated by combining DNA from multiple organisms

Question 6

What is gene cloning?

Answer 6

Isolating and making identical copies of a fragment of DNA

Question 7

What are DNA carries/vectors

Answer 7

vectors that carry and replicate foreign DNA fragments within a cell

Question 8

What are the 5 steps of gene cloning?

Answer 8

• isolate bulk DNA
• isolate specific DNA sequence
• ligation (joining DNA fragment with a self-replicating genetic element
• transform bacteria (force into vector)
• screen bacteria

Question 9

What do vectors do?

Answer 9

Carry and propagate DNA in host cells by autonomous replication

Question 10

How is bacteriophage lambda used as a vector?

Answer 10

• contains a region of DNA that is not required for survival or replication
• this can be taken and replaced by foreign DNA
• when lambda lyses within a bacterium it will released particles each carrying a copy of the foreign DNA

Question 11

What is a bacterial circular plasmid vector?

Answer 11

Supercoiled, double-stranded DNA circles that exist independent of the bacterial genome and can carry foreign DNA

Question 12

How is a vector joined with foreign DNA?

Answer 12

• restriction enzyme creates a sticky 5’ overhang
• the foreign DNA is cleaved with the same enzyme to produce a compatible sticky overhang
• these reanneal with DNA ligase and a recombinant plasmid is formed

Question 13

How is the recombinant plasmid introduced into bacteria?

Answer 13

• treat the bacteria with CaCl2 and heat to make them leaky
• Those that take up the plasmic will now have antibacterial properties so the bacteria can be treated with ampicillin to remove those that haven’t taken it up

Question 14

How are eukaryotic genes cloned?

Answer 14

• reverse transcriptase synthesised cDNA using a primer that binds the poly(A) tail of mRNA
• RNAse H digests the mRNA to form a single strand of DNA
• the ssDNA forms a hairpin that primes DNA strand synthesis and DNA polymerase is added to make dsDNA

Question 15

Why can mature mRNA NOT be used for cloning?

Answer 15

It is not double stranded

Question 16

What is the role of the polymerase chain reaction?

Answer 16

• allows selective synthesis of a gene sequence using prior knowledge of the gene sequence to design ssDNA primers
• PCR amplification Is exponential

Question 17

What is taq DNA polymerase

Answer 17

• DNAp with enhanced stability at high temperatures
• can withstand repeated heating and cooling without denaturing
• lacks 3’-5’ proof reading

Question 18

What is the Tm?

Answer 18

The temperature where 50% of primer is annealed to the template

Question 19

What happens if the primer anealing temperature is incorrect?

Answer 19

• too high = no annealing, no product

- temp too low = mismatches can occur and non-specific products are created

Question 20

What are the 3 parts of the PCR amplification cycle?

Answer 20

denaturation, primer annealing and extension

Question 21

What happens at 95 degrees, 60 degrees and 70degrees in the PCR amplification cycle?

Answer 21

95 - heat denatures dsDNA h-bonds to make ssDNA templates
60 - primers anneal
72 - optimal temperature of taq polymerase DNA synthesis

Question 22

How many times is the PCR amplification cycle repeated?

Answer 22

25-35 times

Question 23

What are the long product and desired produced in PCR?

Answer 23

Long = doesn't have the required 3' end as nothing stops the primers from synthesising along the entire strand 
Desires = in the next PCR step, the products from step 1 hybridize  to the strands and help to form a defined end.

Question 24

Name three uses of PCR?

Answer 24

• gene cloning
• viral screening
• forensics/DNA fingerprinting

Question 25

What is DNA sequencing?

Answer 25

• determines the order of bases in a DNA sequence

- can screen of gene mutations and sequence variants

Question 26

What is sanger sequencing?

Answer 26

• uses di-deoxyribonucleotides to obtain the sequence

- no 3’OH group means it can no longer form a phosphodiester bond and synthesis ends

Question 27

What is sanger DNA sequencing using ddNTPS? (use CTP as an example)

Answer 27

• The primer binds and synthesises DNA
• Whenever a G is encountered, either a dCTP or a ddCTP is incorporated
• when a ddCTP is selected, chain termination occurs
• this reaction can be set up for each dNTP and the sequence can be found by where the chain is stopped each time

Question 28

What is bioinformatics?

Answer 28

The combination of computer science, statistics and maths to analyse biological data

Question 29

What is comparative genomics? Give examples.

Answer 29

• comparing whole genome sequence of different organisms
• human/chimp
• human brain evolution

Question 30

What are SNPs?

Answer 30

• any base variation between 2 or more individuals

- can be inherited in blocks known as haplotypes

Question 31

What do genome-wide association studies on haplotypes show?

Answer 31

• most haplotypes will segregate randomly but some will be found more frequently in individuals with specific diseases