L24- Molecular techniques

Question 1

why do we use restriction analysis?

Answer 1

• To investigate the size of DNA fragments e.g. small deletions
• To investigate mutations e.g. Sickle cell disease
• To investigate DNA variation e.g. DNA fingerprinting

Question 2

what enzymes are used for restriction analysis

Answer 2

endonucleases

Question 3

what produce endonucleases

Answer 3

bacteria

Question 4

specific restriction endonucleases

Answer 4

recognise and cut specific DNA sequences (phosphodiester bonds) (at restriction sites)

Question 5

restrictriction sites are mostly

Answer 5

palindromes

Question 6

when restriction endonuclease cut at palindromic restriction sites we will ge an

Answer 6

uneven stagger

• producing sticky ends
• 5’ overhang- can be joined back together using ligases

Question 7

restriction endonuclease are a type of

Answer 7

molecular scissors

Repertoire of restrictions endonucleases at our deplorable
e.g. BamHI, EcoRI, Pstl

Question 8

how can restriction sites be predicted

Answer 8

using software

Question 9

what alter restriction sites, meaning endonuclease will be useable to cleave DNSA

Answer 9

mutations

Question 10

gene cloning uses

Answer 10

plasmids e.g. from E.coli

Question 11

outline gene cloning

Answer 11

1. Isolate relevant gene of interest following digestion with restriction enzymes
2. Insert gene of interest into plasmid vector (recombinant DNA molecule)
3. Introduce recombinant DNA molecule into suitable host cell e.g. E.coli
4. Identify and isolate the clone containing the DNA of interest

Question 12

plasmids

Answer 12

• Small circular dsDNA
• Found in bacteria
• Mini chromosomes
• Can transfer to other bacteria
• Often carry antibiotic resistance genes

Question 13

why clone human genes

Answer 13

• Make useful proteins e.g. insulin (put into expression vector and gives us human insulin protein)
• To find out what genes do e.g. HTT
• Genetic screening e.g. Huntington’s, BRCA1/2, CF
• Gene therapy e.g. CF

Question 14

polymerase chain reaction is a method of

Answer 14

amplifying small amounts of target sequences rapidly

Question 15

requirements for PCR

Answer 15

• Thermostable DNA polymerase (Taq)
• Pair of primers (forward and reverse), uniquely defining the region to be copied
• Thermocycler

Question 16

why use PCR?

Answer 16

• Amplify specific DNA fragment
• To investigate single base mutations e.g. Tay sachs and sickle cell
• To investigate small deletions or insertions
• To investigate variation, genetic relationships e.g. DNA profiling

Question 17

process of PCR simple

Answer 17

• Denaturation
• Annealing stage
• Extending stage

Question 18

denaturation

Answer 18

(95 degrees): DNA denatures (breaks H bonds and becomes ss)

Question 19

annealing

Answer 19

(50-56 degrees) - specific primers added bind to complementary sequence

Question 20

extending

Answer 20

(72 degrees): Taq polymerase added and DNA synthesis

Question 21

PCR and copying of DNA is

Answer 21

exponential increase in DNA

Question 22

primers are included in

Answer 22

the end product

Question 23

DNA electrophoresis

Answer 23

DNA is negatively charged and will move towards the anode if placed in an electric field- DNA fragments can be separated on the basis of size (or shape).

Question 24

what happens to DNA before electorphoresis

Answer 24

• cut by restriction endonuclease- only specific genes being look at

Question 25

DNA electrophoresis separates DNA on the basis of

Answer 25

size (or shape)

Question 26

requirements of gel electrophoresis

Answer 26

- Gel- a matrix that allows separation of DNA fragments - Buffer- allows charge on the DNA samples across the gel - Power supply- generates charge difference across the gel - Stain- to identify the presence of the

Question 27

protein electrophoresis

Answer 27

- Proteins can be separated based on size, shape or charge

Question 28

why can proteins be separated using electrophoresis

Answer 28

Proteins are charged molecules and will move towards the anode or the cathode if placed in an electric filed. e.g. serum proteins

Question 29

if we want to separate the proteins just on the basis of size

Answer 29

we add detergent SDS (removes charge)

Question 30

adding detergent to proteins

Answer 30

o Makes a folded protein in a linear polypeptide chain

Question 31

requirements of gel electrophoresis

Answer 31

* Gel- a matrix that allows separation of the protein sample * Buffer- maintains charge on the protein samples * Power supply- generates charge difference across the gel * Stain – to identify the presence of separated proteins

Question 32

when a protein electrophoresis is finished, what can be used to identify unknown proteins

Answer 32

Mr standards- to compare size

Question 33

protein identification

Answer 33

proteomics

Question 34

proteomics example

Answer 34

* Digest proteins with trypsin * Perform mass spec * Generate list of peptide sizes * Use database of predicted peptide sizes for known proteins to identifying the specific protein

Question 35

immunoassays

Answer 35

use of antibodies in the identification of protein

Question 36

antibodies have unique structures which recognise specific antigens

Answer 36

paratopes which recognise epitomes on an antigen

Question 37

types of antibodies

Answer 37

polyclonal | monoclonal

Question 38

polycolonal

Answer 38

* Produced by many b lymphocytes * Lots of diff antibodies which recognise diff epitopes on the same antigen * Most commonly used in diagnosis

Question 39

monoclonal

Answer 39

• Recognise a single epitope on a single antigen

Question 40

how can we use antibodies (name 4 techniques)

Answer 40

- Western blotting - Southern blotting - Northern blotting

Question 41

Weston blotting allows

Answer 41

identification of particular proteins from mixture of diff proteins

Question 42

ELISA can be

Answer 42

- Indirect - Direct - Sandwich

Question 43

ELISA used to

Answer 43

- Measure the conc of proteins in solution: o Insulin o Cortisol o TSH ‣ The more antibody that binds the more protein present

Question 44

outline how indirect ELISA works

Answer 44

1) Antigen coated to well 2) specific antibody binds to antigen 3) enzyme- linked antibody binds to specific antibody 4) Substrate is added and converted by enzyme into coloured produced, the rate of colour formation is proportional to the amount of specific antibody

Question 45

enzyme assay

Answer 45

way of calculating rate of an enzyme catalysed reaction

Question 46

how do enzyme assays work

Answer 46

Can measure rate for activity of an enzyme- by adding substrate and measuring conversion of substrate to product over time = rate

Question 47

continuous assay

Answer 47

- Spectrophotometer | • Chemoluminescence

Question 48

discontinuous assay

Answer 48

* Radioactivity | * Chromatography

Question 49

measurement of enzymes can be used to diagnose

Answer 49

metabolic disorders and in diagnosing disease

Question 50

clinically important serum enzymes for liver disease

Answer 50

aspartate transaminase alanine transaminase

Question 51

clinically important serum enzymes for pancreatisis

Answer 51

amylase and lipase

Question 52

clinically important serum enzymes for liger damage (increased by alcohol)

Answer 52

Y-glutamyl transferase

Question 53

clinically important serum enzymes for bone disroders

Answer 53

alkaline phosphatease

Question 54

clinically important serum enzymes for bone disorders

Answer 54

alkaline phosphatase

Question 55

CK-1

Answer 55

brain

Question 56

CK-2

Answer 56

BM (myocardium)

Question 57

CK-2

Answer 57

MM- skeletal

Question 58

gold standard for diagnosis of an MI is the measurement of

Answer 58

cardiac troponin I (cTnl) by ELISA