Mol Bio

Front 1

Average length of DNA fragments

Back 1

1) x the frequencies in decimals

2) = fraction (bottom no. / top no.)

3) round up that number to get average length in bp

Front 2

Chimeric protein

Back 2

Proteins created through the joining of genes that originally coded for separate proteins

Front 3

2-primer mutagenesis: why use restriction enzyme DpnI?

Back 3

To remove the parent template DNA

Front 4

Why use alanine as the aa substitute during scanning mutagenesis?

Back 4

Non-bulky, chemically inert, methyl functional group that mimics the secondary structure preferences that many other aa’s possess

Front 5

Why is non-degenerate saturation mutagenesis better than degenerate?

Back 5

Most efficient as only uses 20 codons, 1 for each of the 20 aa’s. No protein library bias, no truncation, maximises sequence space.

Front 6

Phage display: Why does gene of interest need to be fused in frame with gene encoding the phage coat protein?

Back 6

To avoid frameshift mutations affecting the C terminal protein and resulting in a non-functional gene

Front 7

NG pyrosequencing: How is dNTP detected?

Back 7

Specific primer annealed to template DNA and extended with DNA polymerase, 1 nucleotide is washed over template DNA at a time and if complementary, it incorporates, and 1 pyrophosphate is lost and converted in to a light signal via ATP synthesis and luciferase.

Front 8

Techniques for working out the function of a gene of interest?

Back 8

Random mutagenesis by PCR

Cassette mutagenesis

Site directed mutagenesis

Front 9

Why should specific activity of your protein increase during protein purification?

Back 9

Amount of protein (mg) is typically less but the rate of reaction stays the same (or May increase due to less interference or removal of inhibitors)

Front 10

Stock volume calculation

Back 10

V1 = (V2xC2) / V1

1M = 1000 mM

Front 11

Probability of a restriction site being cut by 2 enzymes calculation

Back 11

1) what bases do we need for the 2 restriction sites to be the same

2) frequency of these bases as a fraction

3) x the fractions e.g. 1/4 x 1/4 = 1/16

Front 12

How is ATP used in ligation?

Back 12

Ligase + ATP = Ligase-ATP + PPi

AMP + phosphate = product-ligase + AMP

Front 13

What would you substitute for glutamine?

Back 13

- charge: glutamic acid (O rather than NH2)

+ charge: lysine (no C=O and 2 extra CH2)

Hydrophobic: methionine (similar length; 2 CH2)

Front 14

What would you substitute for glutamine?

Back 14

- charge: glutamic acid (O rather than NH2)

+ charge: lysine (no C=O and 2 extra CH2)

Hydrophobic: methionine (similar length; 2 CH2)

Front 15

Megaprimer mutagenesis: what cycle is first to generate correct length product with mutation in both strands?

Back 15

Cycle 3

Front 16

Why is TaqMan better than SYBR green staining in QPCR?

Back 16

SYBR green binds to any, including non-specific ds DNA and so false positives may be generated. TaqMan is more specific to the gene of interest

Front 17

Why is TaqMan better than SYBR green staining in QPCR?

Back 17

SYBR green binds to any, including non-specific ds DNA and so false positives may be generated. TaqMan is more specific to the gene of interest

Front 18

What factors must be optimised to max production of a soluble, recombinant protein in E.coli?

Back 18

Temperature, induction point and culture time

Front 19

What is the need for activity assays after each column during conventional chromatography?

Back 19

Ensure protein activity isn’t lost by denaturation over time or if target protein is lost during previous elation, those that lose activity can be excluded from further columns

Front 20

2 enzymes that make both 5’ and 3’ sticky ends of DNA blunt?

Back 20

SmaI and klenow fragment

Front 21

Methods to identify aa in active site responsible for difference specificities?

Back 21

Sequence homology - identifies the region of the protein responsible for specificity.

Alanine scanning - replace each aa with alanine and examine the effect on protein and identify key aa’s.

Saturation mutagenesis - introduces mutations in to a single or set of codons and creates a protein library which can be screened for proteins with required activity

Front 22

Threonine substitutions?

Back 22

- charged: aspartic acid (H and C=O instead of CH3)

+ charged: lysine (NH3+ rather than OH and CH3)

Hydrophobic: valine (same length, only differs by 1 CH3 which is an OH)

Front 23

Features of DpnI

Back 23

1) recognises methylated DNA only e.g. bacterial DNA and not PCR synthesised DNA

2) 4 base recognition sequence which cuts plasmid efficiently

3) recognises small, frequent restriction sites

Front 24

Phage display library

Back 24

Many variants of 1 protein which is screened for required activity

Front 25

Phage display library

Back 25

Many variants of 1 protein which is screened for required activity

Front 26

A) how many different clones theoretically in the library?

B) how much seq space can library occupy?

Back 26

A) e.g. NNK = 32 codons ^7 (number of positions saturated)

B) (1x10^10 / 32^7) x 100 = %

Front 27

5 key features that prokaryotic expression vector must have (E.coli)

Back 27

Promotor, RBS, fusion domain, gene and terminator

Front 28

Affinity tags used in affinity purification

Back 28

Maltose binding protein, glutathione S-transferase, His6 Tag

Front 29

Anion or cation exchange?

Back 29

If pH is > pI (more basic), H+ has been removed (NH3+ -> NH2 and COOH -> COO-), protein is - and sticks to + column.

pI (isoelectric point) is the pH at which the molecule has no net charge and is neutral.