Essay #1 Explain how RFLP analysis has facilitated the process of DNA fingerprinting.

[6] The presence of RFLPs in each individual means that digesting the DNA of each individual with the same restriction enzymes [1/2] o will result in the formation of different numbers and sizes of restriction fragments. [1/2] o As different individuals will have different RFLP variants[1/2] o Due to differences in numbers of restriction sites[1/2] o Or differences in numbers of tandem repeats[1/2]

A DNA fingerprint is the unique pattern of restriction fragments generated by a restriction digest of an organisms genome [1/2] o Identified by Southern Blotting/ nucleic acid hybridisation or by PCR[1/2] These fragments are then separated by gel electrophoresis, denatured and transferred to a nitrocellulose membrane[1/2] o And incubated with radioactively labelled probes complementary to the RFLP loci to be analysed[1/2] The DNA fingerprint can then be analysed/ compared to that of other individuals[1/2] DNA fingerprinting can be used in forensic analysis/ solving criminal cases[1/2] o Or paternity studies[1/2] o Or homology studies[1/2]

Outline how RFLP analysis has facilitated the process of disease detection. [5] RFLPs can be used as genetic markers to detect the presence of disease causing alleles. [1/2]

If the disease causing gene has not been identified and sequenced, an RFLP locus that is tightly linked to the disease causing gene can be used. [1/2] o Crossing over between the RFLP locus and gene is unlikely in gamete formation [1/2] o Thus, the RFLP variant and the associated disease causing allele will be very likely to be inherited together [1/2] o Detection of the RFLP variant means that it is very likely that an individual possesses the disease causing allele as well. [1/2] If the mutation that causes the disease also results in a gain/ loss of a restriction site, [1/2] o The number and size of restriction fragments formed by digestion of the disease causing allele and the normal allele will be different. [1/2] These fragments are then separated by gel electrophoresis, denatured and transferred to a nitrocellulose membrane[1/2]

o And incubated with radioactively labelled probes complementary to the

RFLP loci to be analysed[1/2] Each allele will have a different band pattern that can be used for the identification of the disease causing allele. [1/2] Explain how RFLP analysis can be used to detect the presence of the mutated -globin gene that causes sickle cell anaemia. [7]

Sickle cell anaemia is caused by a single-nucleotide substitution in the beta-globin gene [1/2] o (A T) [1/2] o This results in the loss of a MstII restriction site in the sickle cell allele [1/2] o Thus, the sickle cell allele will only possess 2 MstII restriction sites while the normal globin allele will possess 3 MstII restriction sites [1/2] DNA from the individual is digested with MstII, [1/2] o Then separated by gel electrophoresis [1/2] o And transferred to a nitrocellulose membrane by Southern blotting. [1/2] o The membrane is then incubated with a radioactively labelled probe [1/2] complementary to the beta-globin gene region that includes the nucleotide substitution [1/2] If the normal globin allele is present, digestion of the MstII RFLP variant in the normal globin gene would result in the formation of 2 smaller fragments [1/2] o Thus, the probe hybridizes with 2 small fragments and two bands containing smaller fragments are visualised [1/2] If the sickle cell allele is present, digestion of the MstII RFLP variant present in the sickle cell globin allele would result in the formation of 1 large fragment. [1/2] o Thus, the probe hybridises with 1 large fragment and one band containing the large fragment is visualised. [1/2] Thus, any individual whose restriction fragment pattern shows the presence of the band containing the large fragment would contain the sickle cell allele. [1/2]

Explain how RFLP analysis has facilitated the process of linkage mapping. [6] What linkage mapping is + how linkage mapping is carried out A linkage map is an ordered list of genes or genetic markers [1/2] o that includes the relative distance between them. [1/2]

The order and relative distances between genes and/or genetic markers is based on their recombination frequencies [1/2] Many genetic crosses are carried out to determine the recombination frequencies between genes and/or genetic markers [1/2] o The more often crossing over occurs between genes and/or genetic markers, the further apart they are [1/2]

How RFLP analysis helps in linkage mapping Thousands of RFLPs have been discovered and mapped to individual chromosomes [1/2] o These RFLPs serve as genetic markers in linkage mapping [1/2] o RFLPs also provide an increased number of genetic markers other than gene markers [1/2]

As RFLPs are usually found in non-coding regions, [1/2] o The use of RFLPs as genetic markers allows the mapping of noncoding regions and [1/2] o Of regulatory sequences [1/2] The length of restriction fragments formed also allows one to estimate the number of nucleotides between genetic markers or genes. [1/2]

Thus, by providing an increased range of genetic markers, the use of RFLPs in linkage mapping has made the process of linkage mapping much easier.