Lecture One : Hybridization and DNA cloning techniques ( not finished)

Question 1

what’s DNA made of?

Answer 1

phosphate group
deoxyribose sugar
phosphate group

Question 2

How many strands does DNA have?

Answer 2

2

Question 3

how many carbons does pentose sugar have?

Answer 3

5

Question 4

How many hydrogen bonds do each base pairings have?

Answer 4

AT - 2

CG- 3

Question 5

whats chromatin?

Answer 5

proteins and DNA that are stacked together

Question 6

minor groove in DNA is _____.

Answer 6

smaller

Question 7

what are Purine bases?

Answer 7

A and G

Question 8

what are pyrimidine bases?

Answer 8

T and C

Question 9

whats a chromosome?

Answer 9

dense packets of DNA by which hereditary information is passed from one generation to the next

Question 10

whats a autosome?

Answer 10

any chromosome other than a sex chromosom

Question 11

whats a Gene?

Answer 11

a sequence of DNA nucleotides on a chromosome

Question 12

whats a Allele?

Answer 12

alternative forms of a gene/marker at a specific genetic location

Question 13

whats a STR?

Answer 13

short tandem repeat

Question 14

whats a SNIP?

Answer 14

single nucleotide polymorphism

Question 15

How do we select the desired target region?

Answer 15

hybridisation

Question 16

How do you amplify and purify the desired target?

Answer 16

1. DNA cloning

2. Polymerase chain reaction (PCR)

Question 17

what is hybridisation?

Answer 17

two strands of DNA joining together

Question 18

How can you denature DNA?

Answer 18

1. Heating the DNA

2. Exposure to polar chemicals such as formamide or urea

Question 19

does denaturing DNA do?

Answer 19

breaks it apart into single strand does not damage the DNA

Question 20

what is Re-forming of the DNA strands known as?

Answer 20

nucleic acid annealing or hybridization.

Question 21

what can happen when annealing occures?

Answer 21

homoduplexes or heteroduplexes

Question 22

whats a heteroduplexes?

Answer 22

uplex where base pairing is not perfect across the full

length of a fragment, or when the DNA from each strand is from two different sources.

Question 23

what can DNA pair with?

Answer 23

DNA, RNA and oligonucleotide sequences.

Question 24

whats oligonucleotide?

Answer 24

short synthesised, single stranded DNA molecule, ~50 nucleotides long also sometimes referred to as a probe.

Question 25

oligonucleotide is also known as a ____.

Answer 25

probe

Question 26

what makes a DNA duplex stable?

Answer 26

bases matching between the two molecules.

Question 27

when is stability fo a DNA duplex not affected?

Answer 27

short non complimentary regions don't affect the overall stability

Question 28

what is stringency?

Answer 28

conditions which will affect weather two strands anneal

Question 29

what happens when the conditions of stringency are too low ?

Answer 29

conditions too low, probe/DNA will anneal to non complimentary regions

Question 30

whats the perfect temperature for annealing?

Answer 30

temperature just below the melting temperature (T ) of the DNA or the oligonucleotide being used.

Question 31

what happens when temperature is above Tm?

Answer 31

fully based paired hybrids may be unstable.

Question 32

what happens when temperature below 5°C Tm?

Answer 32

mis-paired hybrids may be stable.

Question 33

what alters alter hybridization stringency?

Answer 33

temperature and salt concentration

Question 34

what will high salt concentration do to stringency?

Answer 34

decrease stringency

Question 35

what will low salt concentration do to stringency?

Answer 35

achieve high-hybridization stringency.

Question 36

what are the two categories of Hybridization assays?

Answer 36

standard assay, reverse assay

Question 37

whats a standard assay?

Answer 37

Labeled probe and unlabeled test sample bound to a solid support.

Question 38

what is a reverse assay?

Answer 38

Unlabeled probe bound to a solid support and labeled test sample

Question 39

whats microarray hybridization?

Answer 39

Uses the reverse assay method, numerous hybridisation reactions happen at the same time.

Question 40

How is microarray hybridisation done?

Answer 40

1. solid support used to create loads of features on a grid 2. Each feature/square contains identical unlabeled oligonucleotides (probe) 3. A test sample containing heterogeneous labelled DNA (or RNA) fragments. 4. After washing the microarray hybridization signal is read using a laser.

Question 41

what do the results of microarray hybridisation show?

Answer 41

strong hybridization signal would indicate a lot of complementary sequences between the test sample and the probe in that feature (square on the grid).

Question 42

can microarray hybridisation be done on RNA?

Answer 42

yes

Question 43

what are the 5 Hybridization methods in Forensic Science?

Answer 43

1. SLP/MLP identification of RFLPs using Southern Blot technique 2. Quantification of DNA and first forensic tests using reverse/dot blot technique. 3. DNA-DNA hybridization in the polymerase chain reaction. 4. SNP identification using hybridization 5. Microarray hybridization for biological fluid identification

Question 44

who developed the southern blott technique?

Answer 44

Edwin Southern in 1973

Question 45

what happens at the start of a southern blott technique? step 1

Answer 45

- DNA is digested with restriction endonucleases - Fragments separated by electrophoresis - Target fragments are identified using labelled probe.

Question 46

what happens to the gel in the southern blott technique? step 2

Answer 46

Gel is treated with alkali, this denatures DNA, its then transferred to a nitrocellulose or nylon membrane.

Question 47

what is the membrane soaked in? SBT step 3

Answer 47

soaked in a solution containing a labelled probe.

Question 48

what happens to the labelled probe? SBT step 4

Answer 48

Labelled probe hybridizes to membrane bound DNA fragments, forming heteroduplexes.

Question 49

what can the label be in SBT?

Answer 49

radioactive or fluorescent