10.1 processing DNA

what does the development of polymerase chain reaction enable (PCR)

enables small quantities of dna to be replicated, producing testable amounts to use in analysis techniques

how does PCR replicate dna?

mimics the natural process of dna replication occurring prior to cell division

what are the 3 steps that DNA goes thru in PCR

denaturing: 2 strands are separated

annealing: short sections of dna primers are bound to the seperate strands

extension: short sections of dna are extended to produce longer strands

explain denaturing

heat is used to break the hydrogen bonds holding the 2 strands together.

explain annealing

temperature is decreased

primers bind to the single dna strand

explain the dna primers

they are complementary to either end of the section of dna to be copied

explain extension (elongation)

DNA polymerase is used to join new complementary nucleotides where the primers begin (5 prime to 3 prime in that direction.)

why is there a large amount of dna polymerase used in this process

dna polymerase attaches to the double stranded dna before denaturation, this process occurs at 94-96 degrees celsius which destroys dna polymerase. therefore more dna polymerase has to be added.

what is gel electrophoresis

a technique that is able to seperate dna strands based on their lengths.

what occurs to dna samples prior to gel electropherosis

restriction enzymes cut the dna at specific sites

explain the process of gel electrophoresis (5)

1. dna pieces are placed in wells in a semi-solid gel that is in a solution of electroclyte

2. in solution there are electrodes

3. the negative electrode is closest to the wells (dna) the positive is on the opposite end

4. when a current passes thru, the negatively charged dna moves to the positive electrode

5. the smaller dna pieces move faster than larger ones, and are located closer to the positive electrode when current stops.

what is the type of pattern produced and its name

similar to barcodes. banding pattern = an individuals dna profile/dna fingerprint

how is dna accurately placed in solution and what are its advantages

micropipette, this reduces any chances of cross contamination due to disposable tips

what is a dna ladder

contains segments of dna with known lengths, results from gel electrophoresis are compared to the dna ladder to determine the length of dna strands

how are the dna samples visualised after gel electrophoresis

ethidium bromide, methylene blue, or dna probes

what is dna sequencing

determination of precise order of nucleotides in a sample of dna

what happens to nucleotides when dna forms

each nucleotide loses 2 phosphate groups and the sugar molecule loses a hydrogen atom from the hydroxy group when it bonds to the phosphate group of an adjacent molecule

who’s method is used for the determination of dna sequencing

sangers method

how does Sangers method work

nucleotides that lack the OH group are added to a growing strand. this stops the elongation of the sequence because there is no OH group for the next nucleotide to attach to.

what is the name of nucleotides with no OH group

dideoxyribonucleotides (ddNTPs)

what occurs to the sequence after the elongation stops

different lengths of dna are created, separated through dna electrophoresis. knowing which base was added to create each length allows scientists to determine the order of nucleotides

what can dna sequencing be used for

to identify muttions/compare dna:

sickle cell anaemia, cystic fibrosis, some forms of cancer.

maternity/paternity tests

also to compare species for evolutionary changes

what are ethical considerations when using genetic info

autonomy, confidentiality, equity, and privacy