Unit 3 - Biotechnolgy pt 2 (DNA Sequencing)

Question 1

Polymerase chain reaction

Answer 1

PCR is a powerful process that can greatly increase the number of copies of DNA from one biological sample within a few hours

Question 2

PCR facts (5)

Answer 2

1) only specific region is duplicated
2) plasmids not used bf 1980’s
3) PCR copying DNA directly; while plasmids indirect
4) is fast, reliable and inexpensive
5) PRC is dome in vitro (occurs in small test tube)

Question 3

PCR three steps

Answer 3

denaturation
annealing
elogation
theses steps r repeated in many cycles usually 30 to 4o

Question 4

PCR material (4)

Answer 4

• DNA template sequnce
• taq DNA polymerase
• nucleotides
• primers

Question 5

Denaturation

Answer 5

double-strand DNA mol is denatured or separated into its two single strands. this provides ss temple for the next step

Question 6

Denaturation how? (HEAT)

Answer 6

DNA strand heated to 94-96c for 20 to 40s
this causes hydrogen bonds holding the two stand together to break

Question 7

Annealing

Answer 7

• mixture is cooled back down to 50-65c for 20 to 40z
• reaction is cooled so the DNA primers can bind to their complementary sequences at the two ends of the target sequence of single stranded template DNA

Question 8

DNA primer

Answer 8

a short single-strand DNA sequence, easily synthesized in a laboratory that is complementary to the sequence at one end of the other target sequence.

Question 9

Elongation

Answer 9

mixture heated back to 73c so a DNA polymerase known as Taq polymerase can extend the primers to synthesize new strands of DNA

Question 10

taq polymerase

Answer 10

is extracted from the bacterium thermus aquatics. which is found in hot springs and can with stand high temps unlike DNA polymerase

Question 11

cycle 2 vs cycle 3

Answer 11

= 4 mols
= 8 mols
cycle is repeated to 30 to 40 times, resulting in a high number of copies of a specific target sequence

Question 12

how long does it take

Answer 12

only 5 mis millions of copies of DNA sequence can be synthesized in less than 2h

Question 13

PCR efficient process

Answer 13

used almost exclusively to amplify a target DNA sequence. only a v small amt of biological sample is required for a PCR such as a DNA form a hair follicle, tiny blood spatter, or fish tissue

Question 14

Once DNA of interest has been amplified

Answer 14

using PCR it is analyzed using a diff method

Question 15

Gel electrophoresis

Answer 15

used to seperate DNA fragments according to their chemical and physcial properties (IE. mass and electrical charge)

Question 16

when is gel electrophoresis useful

Answer 16

when trying to isolate a gene that has already been excited by using a restriction enzyme and amplified

Question 17

Gel electrophoresis is like molecular sieve

Answer 17

used to separate the desired excised gene from other pieces of DNA

Question 18

what is needed in gel E (3)

Answer 18

1) electricity - (+) and (-) anodes
2) buffer solution - contains salts to conduct electricity
3) gel - submerged into buffer; porous matrix like support fro DNA to weavee way through

Question 19

Step 1 of Gel Electrophoresis

Answer 19

samples of different sizes of DNA + to gel in pre-formed wells at one end with two chemical added to sample
- negatively charged dye turns sample blue
- Ethidium bromide

Question 20

ethidium bromide

Answer 20

sticks to DNA and fluoresces under UV light

Question 21

Step 2 of Gel Electrophoresis

Answer 21

electricity runs between anode and cathode
- DNA travels in their respective land towards (+) cathode

Question 22

Step 3 of Gel Electrophoresis

Answer 22

smaller DNA fragments experience less resistance = travel further
-blue dye helps see how far DNA travles

Question 23

large DNA fragments Vs. small

Answer 23

move slower through gel then small frags. this means that smaller frag move further in the gel

Question 24

Step 4 of Gel Electrophoresis

Answer 24

gel is removed from buffer and exposed to UV light

Question 25

molecular marker

Answer 25

containing fragments of known size is run on the same gels as the digested DNA. reachesers can then determine the size of the unknown fragments by comparing mm

Question 26

what is the main function of restriction enzymes

Answer 26

to cut DNA at specific sites within DNA

Question 27

what enzyme catalyzes the joining of fragments to form recombinant DNA

Answer 27

DNA ligase

Question 28

name of process that introduces DNA into bacterial cells

Answer 28

transformation

Question 29

why r bacteria the most common hosts used in gene cloning

Answer 29

bacteria grow quickly in large amts and r inexpensive to maintian

Question 30

Paternity testing and forensics

Answer 30

The technique of gel electrophoresis has wide applications. Two typical examples of applications of the technique are paternity testing and in DNA fingerprinting in forensic science. First, scientists amplify specific sequences from more than one DNA sample. The amplified sequences are then cut using the same restriction enzymes. Identical samples of DNA will be cut in the same places producing identical fragment sizes. The greater the number of differences between fragment sizes the less related the individuals must be.

Question 31

STR analysis in a parentage dispute:

Answer 31

A simplified example of the use of STR (short tandem repeat) analysis in a parentage dispute (using 3 STR loci). Half of the STR bands come from the mother and half from the father. Some bands are inherited from both the mother and the father. If a child's DNA profile contains bands that are present in neither the mother nor the supposed father's DNA profiles, then the supposed father is not the child's natural father. Should the alleged father in this example be instructed to pay child support

Question 32

Restriction Mapping

Answer 32

Desired genes can be inserted into bacterial plasmids for genetic engineering. To do this in bacteria, desired genes can be added to plasmids. When plasmids are inserted into bacteria they will express the genes and make the new protein. To design a plasmid carrying the desired genes and promoters, it is useful to map where specific restriction enzymes are able to cut plasmids from a plasmid library.

Question 33

DNA Sequencing: Chain Termination Method

Answer 33

The Sanger method of DNA sequencing is the foundation of many modern-day sequencing technologies. To sequence a DNA fragment, it is first made into a single strand. The fragment is then amplified using PCR, containing low concentrations of chain-terminating dideoxynucleotides (ddNTP) (each is also labelled with fluorescent dyes). Each time a fragment is copied, if a dideoxynucleotide is incorporated then the synthesis of the strand stops creating a shorter fragment corresponding to the random insertion of that particular letter in the sequence. Using gel electrophoresis, the fragments are arranged by size and the sequence is read from the shortest to longest fragment.

Question 34

PCR is run four separate times with the target single stranded DNA for sequencing.

Answer 34

In the PCR include: DNA oligonucleotides (DNA Primers) Thermostable DNA Polymerase (Taq Polymerase) A high concentration of ALL FOUR nucleotides dATP AND dCTP AND dGTP AND dTTP A small concentration of ONE dideoxynucleotides ddATP then ddCTP then ddGTP then ddTTP

Question 35

Automated DNA Sequencing

Answer 35

A few simple advancements have made the Sanger method much simpler and more efficient. The first major advance was developing dideoxynucleotides with fluorescent dyes attached. These fluorescent tags can be detected with a laser. This means that all four types of dideoxynucleotides can be used in the same PCR cycle. The resulting fragments can be run through a single tiny gel electrophoresis capillary tube with a laser pointed at the bottom. As fragments exit the tube, the laser causes each emerging fragment to fluoresce a particular colour to indicate the last letter encoded.

Question 36

Polymerase Chain rxn

Answer 36

process used to make huge number of copies of a DNA sequnce in lab