Chapter3 Lec3

Question 1

Previously genomes were only reflected by what?

Answer 1

Phenotypes

Question 2

What does it mean now that we can interrogate DNA sequences directly?

Answer 2

It means that any features of DNA that vary among individuals can serve directly as markers.

Question 3

What were the first genetic markers based directly on DNA sequences, rather than on phenotypic traits? And provide it’s function.

Answer 3

Restriction fragment length polymorphism (RFLPs)- is an enzyme that cuts DNA at specific sequence, typically 4,6, or 8 bp ,which are mainly palindromic(meaning that the seq is equal to it’s reverse complement; EcoRI recognition)

Question 4

Discuss each of the other useful types of markers and what they include.

Answer 4

(1) Variable number tandem repeats(VNTRs)
- also called minosatellites
- contains regions/motif seq of 10-100bp long
- repeated a variable no.of times, same seq,different no.of repeats.
- in an individual, VNTRs with same repeat motif may appear once or several times, with different lengths on different chromosomes.
- inheritance of VNTRs can be followed in a family and correlated with a disease phenotype like any other trait
- VNTRs were the 1st genetic sequence data used for personal ID - thus, genetic fingerprints- paternity and criminal cases.

(2) Short tandem repeat polymorphisms (STRPs)
- called microsatellite
- are regions/motifs sequence of 2-5 bp long
- repeated many times typically 10-30 consecutive copies
- they have several advantages as markers over VNTRs, one of which is a more even distribution over the human genome.
- there is no reason why STRs need to lie within expressed gene regions; usually they do not(except for CAG repeats in the gene for Huntington’s disease and certain other disease gene)

Question 5

Name the applications of markers in several different fields, and what they do?

Answer 5

(1) medical- use of tissue typing to identify compatible donors for transplants, detection of disease susceptibility and prediction individual drug-response variation (e.g.pharmacogenomics)
(2) anthropology- use them to trace migrations and relationships among populations
(3) previously, mtDNA or haplotypes were used as markers, but now entire genomes are also being used and replaced older techniques.

Question 6

Suppose you cut a long DNA molecule- e.g.the DNA of an entire chromosome- into fragments of convenient sizes for cloning and sequencing requires additional ___to report the _______,so that the entire chromosome can be ______using the ____.

Answer 6

Maps, order of fragments, reconstructed, fragments.

Question 7

Cutting DNA with a REase produces_____.cutting the SAME DNA with other REases, with different recognition sequences will produce _____. From the sizes of the fragments produced by individual REases and in combination, it is possible to construct a_____ ,which indicates the ___ and ___between the _______.

Answer 7

A set of fragments. 
Overlapping fragments 
Restriction map
Order
Distance 
RE cleavage sites

Question 8

How does finer dissection of a DNA molecule occur?

Answer 8

Via RE digestion, cloning and sequencing

Question 9

In the past, correlations between chromosomes, Gene’s and DNA sequences were essential for identifying:

Answer 9

(1) the molecular links underlying inherited diseases

(2) sequencing of human genome for example, has changed the situation radically.

Question 10

When did Frederick Sanger win the two Nobel prizes in chemistry and for what?

Answer 10

In 1958 for sequencing insulin protein

In 1980 for sequencing RNA and DNA

Question 11

What were the challenges in the early 1970s with regards to DNA sequencing.

Answer 11

1. Preparing pure samples of SADNESS
2. REs- new discovery; not entirely understood
3. Bacteriophage øX-174 - natural source of 5386 bp of purifiable ssDNA; genome still too long; sequence assembly challenge- sequence relatively longer fragments.

Question 12

Identify and discuss the requirements for Sanger sequencing.

Answer 12

1. DNA polymerase- is a replication enzyme for DNA synthesis
2. Primer- provides free 3’OH group for DNA pol to start synthesis
3. Deoxynucleoside triphosphates(dNTPs)- nucleotides used to form a newly synthesized strand.
4. dideoxynucleoside triphosphates (ddNTPs) - dNTPs that lack free 3’OH, leading to termination of DNA synthesis.

Question 13

Sanger sequencing also forms the basis of the_____.

Answer 13

PCR(Polymerase chain reaction)

Question 14

What is the Maxam and Gilbert chemical cleavage method used for?

Answer 14

To separate cleavage reactions to produce fragments that share the 5’- labelled end, but differ in bases at which specific 3’-cleavage occurs.

Question 15

Give the two disadvantages of Maxam and Gilbert chemical cleavage method.

Answer 15

1. No primed DNA: its applicability is inherently limited to sequences adjacent to restriction sites or other fixed termini
2. Use toxic reagents, notably of hydrazine, a neurotoxin.

Question 16

Provide the automated DNA sequencing.

Answer 16

• Fluorescent dyes- (vs radioactivity)
• Single reaction- (vs four reactions)
• Single-lane during (capillary) electrophoresis (vs PAGE) - ABI and DuPont
• Fixed-point laser scans and ID fragments as they migrate- four coloured chromatogram

Question 17

Why is DNA sequence quality important?

Answer 17

• The phred score (q) - measures sequencing accuracy
• q=20 implies a probability of smaller or equal 1% error per base
• common unit of sequencing costs is US$ per 1000 bases determined to q=20 accuracy - a sequence of 1000 bases with q=20 bases would be expected to contain no more than ten errors.

Question 18

Give the formula of DNA sequence quality.

Answer 18

q= -10 log10(p)