[FMS] MCG - Molecular & Cell Genetics - Manipulation of DNA

Question 1

what do restriction enzymes function as?

Answer 1

Restriction enzymes function as ‘molecular scissors’ to cut DNA

they recognize specific nucleotide sequences in the DNA and cuts both strands of the sugar-phosphate backbone

leaves sticky ends

Question 2

what is a palindrome?

Answer 2

sequence reads the same on both strands in 5’-3’ direction

Question 3

DNA is visualised in gel by adding what?

Answer 3

ethidium bromide which binds DNA and fluoresces when exposed to UV light.

Question 4

what sequence do restriction enzymes form?

Answer 4

palindrome sequence

Question 5

what do plasmids contain that allows replication of the bacterial chromsomes independently?

Answer 5

Plasmids contain an origin of replication that allows replication independently of the bacterial chromosome.

This allows high copy numbers of plasmids, 100-200, to be maintained in each bacteria.

Question 6

what do plasmids contain that allows the selective growth of bacteria that contain plasmids.

Answer 6

Plasmids also contain antibiotic (AB) resistance genes that allows the selective growth of bacteria that contain plasmids.

Question 7

outline DNA cloning steps

Answer 7

To clone a DNA fragment the fragment and plasmid are cut with the same restriction enzyme.

Both plasmid and fragment will have same complementary cohesive ends

Cut DNA fragments and cut plasmid are mixed together

DNA fragments will anneal to cohesive ends in plasmid

DNA ligase will ligate the DNA fragment and plasmid together to form recombinant DNA molecule

Question 8

When cloning eukaryotic genes we need to use ….

Answer 8

mRNA because the introns are removed - cant use genomic DNA with introns because its too large

Question 9

When cloning eukaryotes, you use mRNA instead of genomic DNA because the introns are removed, however you cant clone mRNA so what do you do?

Answer 9

Complementary DNA (cDNA) is a DNA copy of a mRNA, produced using an enzyme called reverse transcriptase.

therefore for eukaryotic cloning, cDNAs are used as the introns are removed and the clone size is smaller

Question 10

compare the origin, structure, and function of cDNA and genomic DNA

Answer 10

ORIGIN:

cDNA (complementary DNA):
- synthesized from messenger RNA (mRNA) through a process called reverse transcription.

Genomic DNA:
- complete set of genetic material present in a cell or organism.

STRUCTURE:

cDNA:
- lacks introns, only contains the exonic sequences
- smaller in size

Genomic DNA:
- coding and non-coding regions.
- larger in size

FUNCTION:

cDNA =
- PCR (polymerase chain reaction)
- gene cloning.

Genomic DNA=
- development, functioning, growth, and reproduction of an organism.
- template for transcription
- essential for inheritance and the transmission of genetic traits from one generation to the next.

Question 11

who was DNA sequencing founded by?

Answer 11

Sanger 1980

Question 12

outline DNA sequencing method

Answer 12

Uses DNA polymerase to copy single stranded DNA

Makes use of ‘dideoxy’ nucleotides which interrupt the ability of DNA polymerase to copy DNA

DNA polymerase cannot continue to extend the chain of nucleotides after incorporation of a dideoxynucleotide

Method known as the dideoxynucleotide chain-termination sequencing

This method can be automated and allows rapid sequencing of large amounts of DNA

Question 13

what do ddNTPs have that make them differnt from dNTPs that doesnt allow DNA polymerase to function as normal?

Answer 13

DNA sequencing uses dideoxynucleotides (ddNTPs) which do not have a –OH on the 3’ carbon

DNA polymerase cannot incorporate any further nucleotides after incorporation of a ddNTP as no 3’ OH to form phosphodiester bond

Question 14

what are the 4 types of dNTPs and ddNTPs

Answer 14

remember, they all function the same way where they ALL LACK -OH ON 3RD CARBON so DNA polymerase cant proceed after they’ve been added into the chain

Question 15

in automated DNA sequencing how can we tell which dNTP and which ddNTP is what?

Answer 15

theyre marked with fluroescent marker and they can be identified by colour and separated on capillary gel

on each ddNTP has a different wavelength

The laser light excites the fluorescent tag on each fragment as it passes

The wavelength of the fluorescence is read as the fragment passes, and the information is recorded.

Question 16

3 types of sequencing technologies

Answer 16

Original radioactive dideoxy sequencing on gels

Fluorescent capillary dideoxy sequencing

Next generation sequencing (NGS)

Question 17

what is PCR

Answer 17

an alternative method to make many copies (clones) of specific DNA fragments without need to use cloning vector

Question 18

3 stages of PCR

Answer 18

denaturation
primer annealing
extension.

^ These are repeated over and over using a thermocycler to amplify the DNA exponentially.

Question 19

the number of copies of a fragment in PCR is multiplied by how much in each cycle?

Answer 19

doubled in each cycle:

2,4,8,16, 32, 64, 128, 256 etc

so to work out how many copies made at end of PCR you do 2 to the power of how many cycles ie after 7 cycles how many copies made in PCR - youd do 2 to the power of 7 to give you 128 copies

Question 20

describe the 3 stages of PCR

Answer 20

Denaturation: the reaction is heated to 95°C to denature the DNA into single strands

Primer annealing: the reaction temperature is reduced to 45-68°C to allow primers to hybridize to their complementary sequences in the target DNA

Primer extension: the reactions temperature is raised to 72°C to allow Taq polymerase to synthesize DNA

^ Amplified DNA fragments are separated by gel electrophoresis

Question 21

what are DNA microarrays

Answer 21

use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample.

Question 22

describe the process of DNA microarrays

Answer 22

• all the mRNA from a sample is converted to cDNA,
• fluorescently labelled, denatured into single strands and used as probe.
• This probe is applied to the array which contains spots where single stranded DNA from each gene in the genome is laid out in an array.

Question 23

how is DNA microarray data read?

Answer 23

the labelled cDNA will bind to the spots on the slides that contain complementary sequences

the spots that bind the labelled will then fluoresce under laser light in a piece of equipment known as a chip reader

the location and brightness of the fluorescent spots on the microarray identify the genes expressed in the test sample and their level of expression