L7,8,9 - Molecular Biology Techniques

Question 1

What does plasmid contain?

Answer 1

Origin of replication, antibiotic resistance genes and multiple cloning sequence (MCS)

Question 2

What does RFLPs stand for?

Answer 2

Restriction fragment length polymorphisms - changes in DNA sequence at a restriction site changes the sizes of restriction fragments

Question 3

Expression vectors

Answer 3

are plasmid cloning vectors designed to allow expression of cloned genes in bacteria with the purpose of producing large quantities of encoded protein

Question 4

Eukaryotic expression vectors

Answer 4

are plasmid cloning vectors designed to allow expression of cloned genes in eukaryotic tissue culture cells

Question 5

In dideoxynucleotide sequencing, what gel is used?

Answer 5

Polyacrylamide gel

Question 6

What is PCR?

Answer 6

It is a powerful technique that has been recently developed to allow selective amplification of specific regions of DNA - make many copies of specific DNA fragments without need to use cloning vectors or restriction enzymes

Question 7

How many steps are involved in PCR and what are they?

Answer 7

3 steps - Denaturation, Primer annealing, Extension (DPAE)

Question 8

Denaturation

Answer 8

temperature: 95 degrees to denature the DNA into single stranded (30 seconds)

Question 9

Primer Annealing

Answer 9

temperature: 58 degrees to allow primers to hybridise to their complementary sequences in the target DNA (30 seconds)

Question 10

Primer extension

Answer 10

temperature: 72 degrees to allow Taq polymerase to synthesise DNA (1 minute)

Question 11

What are the limitations of PCR?

Answer 11

1) some informations about the nucleotide sequence pf that target DNA must be known in order to synthesise primers
2) Minor contaminations of sample DNA from other sources can cause problems
3) PCR cannot amplify long segments of DNA, only those that are relatively short (5-10kb max)

Question 12

Reverse transcription PCR (RT-PCR)

Answer 12

is used to study dene expression by examining mRNA production by cells or tissues

Question 13

Quantitative real-time PCR (qPCR)

Answer 13

allows researcher to quantify amplification reactions as they occur in real time to identify amount of DNA in a sample

Question 14

Nucleic acid hybridisation

Answer 14

it is a method to allow identification of nucleic acids fragments or clones containing specific sequences

Question 15

What is used as probe in nucleic acid hybridisation?

Answer 15

ssDNA

Question 16

DNA probe is labelled by using DNA polymerase to incorporate labelled dNTPs. dNTPs can be labelled using…

Answer 16

1) radioactively using 32P
2) attaching a fluorescent molecule
3) attaching digoxygenin

Question 17

What can Colony Blot hybridisation use for?

Answer 17

• it can be use to identify clones in a cDNA library containing specific DNA sequences
• Grow colonies of bacteria, each derived from single bacteria which contain an individual plasmid clone from cDNA library

Question 18

Types of nuclei acid hybridisation

Answer 18

Colony Blot hybridisation, Southern Hybridisation, Northern Hybridisation, In Situ hybridisation and DNA microarray

Question 19

Southern Hybridisation Blot

Answer 19

Allows identification of DNA molecules containing specific sequences

Question 20

Northern Hybridisation Blot

Answer 20

• Allow identification of RNA molecules containing specific sequences
• Most often used to identify if a specific gene is expressed, i.e. transcribed into mRNA, in a particular tissue or sample

Question 21

in situ hybridisation

Answer 21

• hybridising a probe directly to RNA without blotting

- Probe is hybridised to the mRNA transcript in situ i.e. in the place where the transcript is made

Question 22

DNA microarrays

Answer 22

• They are modern devices which use nucleic acid hybridisation to rapidly measure which genes are expressed in a tissue sample
• uses a reverse approach whereby ALL the mRNA from a sample is converted to cDNA, fluorescently labelled 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
• enables the ability to simultaneously identify all the genes expressed in a particular sample