Molecular Techniques

Question 1

What produce endonucleases

Answer 1

Bacteria

Question 2

What do restriction enzymes do

Answer 2

Recognise and degrade foreign DNA

Question 3

Which electrode do DNA fragments travel toward in electrophoresis and why

Answer 3

Positive as DNA is negatively charged

Question 4

What sized fragments travel the furthest

Answer 4

Small

Question 5

What does DNA gel electrophoresis do

Answer 5

Separate fragment based on size

Question 6

How are genes cloned

Answer 6

Isolate specific gene with restriction enzymes and insert into plasmid vector to place into bacteria to replicate naturally

Question 7

What are plasmids

Answer 7

Small circular DNA

Question 8

Give an example of when human genes are cloned

Answer 8

To make human insulin

Question 9

What enzyme concerts mRNA into cDNA

Answer 9

Reverse transcriptase

Question 10

What disease is currently being treated using gene therapy

Answer 10

Cystic fibrosis

Question 11

What enzyme is used in PCR and why

Answer 11

Taq Polymerase as it can work at higher temperatures

Question 12

What is the first step in PCR

Answer 12

Heat strands to 95 degrees to denature DNA to give single strands

Question 13

What is the second step of PCR

Answer 13

Cool DNA to 55 degrees to allow DNA primers to bind

Question 14

What is the final step of PCR

Answer 14

Heat to 72 degrees to allows taq Polymerase to bind and add nucleotides from the 3’ end.

Question 15

Why is PCR used

Answer 15

Look for mutations, to see if there’s a loss or gain of restriction fragments or to see size of product

Question 16

Why can you use PCR to determine if someone has sickle cell anaemia

Answer 16

In sickle cell the mutation changes a restriction site and so this site will not be cut like it would in normal DNA

Question 17

What does PCR result in

Answer 17

Lots of copies of a specific piece of DNA

Question 18

What is protein gel electrophoresis

Answer 18

Separation of proteins

Question 19

What different types of protein electrophoresis are there?

Answer 19

SDS page, isoelectric focussing and 2D page

Question 20

What is SDS page

Answer 20

When proteins are denatured, given a negative charge and separate due to size as they migrate to the positive electrode

Question 21

What is 2D page separation

Answer 21

Separation of proteins based on size and charge

Question 22

What is isoelectric focussing

Answer 22

Separation of proteins based on charge. Protein migrate until they reach a pH equal to their pI

Question 23

What is proteomics

Answer 23

Protein identification

Question 24

How do you carry out proteomics

Answer 24

Digest protein, perform mass spectrometers and identify protein based on peptide sizes

Question 25

What Techniques uses antibodies instead of DNA hybridisation

Answer 25

Western blotting

Question 26

What is ELISA

Answer 26

A technique to detect protein concentration in a mixture by using antibodies to bind to the protein

Question 27

What is an enzyme assay

Answer 27

A technique used to measure enzyme activity by measuring appearance of product or disappearance of substrate

Question 28

The concentration of which enzyme increases during a heart attack

Answer 28

Creatine Kinase

Question 29

What are monoclonal antibodies

Answer 29

Antibodies that can bind to 1 epitope/protein only

Question 30

What are polyclonal antibodies

Answer 30

Antibodies that can bind to multiple epitope in the same antigen

Question 31

Outline DNA hybridisation

Answer 31

When a double stranded DNA is heated and denatured and then labelled, complementary markers are allowed to anneal to the single strands.

Question 32

Outline southern blotting

Answer 32

Separate DNA using electrophoresis then transfer to membrane, denature and hybridise with fluorescent probes to visualise DNA fragments

Question 33

What is northern blotting

Answer 33

Uses DNA in a method similar to northern blotting but detects RNA so tells you gene expression

Question 34

What is western blotting

Answer 34

Using antibodies to visualise proteins

Question 35

What produces DNA probes

Answer 35

Oligonucleotide synthesiser

Question 36

What are ddNTPs

Answer 36

DNA nucleotides that have 3 phosphate groups and no OH group

Question 37

What happens when ddNTPs are incorporated into the growing chains

Answer 37

They stop the chain from growing as the lack of OH stops phosphodiester bonds being made

Question 38

What technique uses ddNTPs

Answer 38

DNA sequencing

Question 39

What can you do with micro arrays

Answer 39

Investigate many genes at one time

Question 40

Outline micro arrays

Answer 40

You compare gene expression from a healthy and diseased patient by mixing samples with hybridisation probes and look at colour to determine expression

Question 41

What regions are compared during DNA fingerprinting

Answer 41

Minisatellites

Question 42

What are minisatellites

Answer 42

Non coding regions of DNA

Question 43

Why are minisatellites compared for DNA profiling

Answer 43

Everyone has different amount of different minisatellites

Question 44

What is karyotyping

Answer 44

Displaying metaphase chromosomes

Question 45

What is FISH

Answer 45

Making probe which are specific for a whole chromosome or gene so that these areas can be visualised

Question 46

What ethical issues are there with investigating the genome

Answer 46

Potential for terminating babies, can't identify all mutations, can lead to future personal consequences (e.g with genetic diseases you may or may not have children )

Question 47

What are reasons for testing someone's genome

Answer 47

To diagnose conditions, for prenatal screening for pregnancy decision and to see if someone's a carrier

Question 48

What is a primer

Answer 48

Short section of DNA that acts as a starting point for DNA synthesis as Polymerase binds here

Question 49

What is reverse transcriptase PCR

Answer 49

Starting with an mRNA strand, producing the cDNA strand and amplifying it